CN108341882A - Merge the preparation and application of pig antibacterial peptide FPAP recombination yeast bacteria preparations - Google Patents

Abstract

The invention discloses the preparations and application of fusion pig antibacterial peptide FPAP recombination yeast bacteria preparations.Fusion protein provided by the present invention, including pig antibacterial peptide PR 39, pig antibacterial peptide Tritrpticin, pig antibacterial peptide PAMP23 and pig antibacterial peptide PG1；The pig antibacterial peptide PR 39, pig antibacterial peptide Tritrpticin, pig antibacterial peptide PAMP23 and pig antibacterial peptide PG1 amino acid sequence be respectively sequence 1 the 1st 42, sequence 1 the 51st 63, sequence 1 the 72nd 94 and sequence 1 the 103rd 129.Experiments have shown that, the FPAP of the present invention can promote the proliferation of lymphocyte, red blood cell and leucocyte, inhibit the growth of pathogenic microorganisms, promotes the secretion of non-specific antibody (IgG, IgG1, IgG2a) and disease specific antibody, improve the immunocompetence and survival rate of animal.

Description

Merge the preparation and application of pig antibacterial peptide FPAP recombination yeast bacteria preparations

Technical field

The invention belongs to biotechnology more particularly to a kind of systems of fusion pig antibacterial peptide FPAP recombination yeast bacteria preparations Standby and application.

Background technology

Now the limitation aquaculture industry of China main problem that further develops and increase economic efficiency be Animal diseases control, The development and production of animal product Quality advance and high-quality cheap feed.China's export animal product because quarantine and the residual quality of medicine compared with Low, outlet proportion is very low, if pork is more than 230,000 tons, only accounts for 4% or so of the quantum of international trade.Especially Sichuan animal is raised The 70% of the energy feeds raw material such as 90% and corn of the protein raw materials such as the dregs of beans of material, which all relies on, to be externally supplied, this just seriously makes The about raising of the Animal husbandry production development and economic benefit of our province.Therefore, how to be developed using High biotechnology efficiently special Colour biological feed makes full use of local feed resource, reduces the use of antibiosis class element additive in feed, overcomes animal product The residual harm of medicine ensures that the production of the organic animal foodstuff of green, protection people's life are healthy and safe, it has also become the present age, there is an urgent need for solutions Great science and technology certainly and social economy's problem.

Current China's livestock and poultry breeding industry is improved with intensive degree, and livestock and poultry cultivation scale and cultivation density are growing, It is strong under the pressure of the pressure of control more than 30 kinds of infectiousness cause of disease transmission of infection diffusion, especially bird flu and breathing breeding syndrome etc. The outburst of sick successive cycle, diarrhea caused by drug-fast bacteria in animal-breeding, sramana, Escherichia coli, streptococcus, blue otopathy (PRRSV), Various bacteriums, the viral diseases such as circovirus (PCV2), swine fever (CSFV) are that bottleneck limitation is asked in livestock-raising for a long time Topic, the serious development for hindering animal husbandry；Keep antibiotic etc additive very serious in the abuse condition of feed, livestock and poultry Have become with the pollution problem of antibiotic feed additive and restricts China's livestock and poultry breeding industry development level and economic benefit raising Bottleneck.The abuse of fodder antibiotics additive not only improves feeding cost, also leads to the drug resistance of pathogenic microorganism and causes a disease Power is remarkably reinforced, and seriously destroys the microecological balance of animal alimentary canal, remains and is enriched in animal body after long-time service, Livestock and poultry body weakens the general level of the health because of poisonous side effect of medicine, and immune disease-resistance power is remarkably decreased.Such vicious circle, livestock and poultry are various Strong sexually transmitted disease frequently occurs, it is difficult to control and treatment.And on the other hand, the animal products food of medicament residue and getting worse Product safety problem not only directly threatens the health of mankind itself, also counteracts the development of aquaculture.

Animal feed adds antibiotic, and induction germ not only drug resistance is occurred by long-term or inappropriate use, but also dynamic There is medicament residue in produce product, cause serious food safety hazards, damage human health and incur the medicine for the treatment of antibiotic Object fails.The developed regions such as European Union, the U.S. start to limit antibiotic for 2006 comprehensively as feed addictive.China also will be stringent It limits feed and adds antibiotic.Modern farming is badly in need of the noresidue of exploitation substitution conventional antibiotic, non-resistant induction, safe nothing The new bio feed and its additive of pollution.

Antibacterial peptide molecular weight is small, isolates and purifies and examine that there are certain difficulties.At present in scientific research antibacterial peptide it is main come Source is chemical synthesis, but chemical synthesis is of high cost, and amount is small, is unfavorable for large-scale application.So we utilize genetic engineering hand Section, recombination connect multiple antibacterial peptide genes, obtain with the anti-multiple-microorganism of wide spectrum (G+, G-, virus, fungi, parasite) and The efficient recombinant antibacterial peptide molecule of immunoregulatory activity.

Also gradually increase in relation to antibacterial peptide gene and other related gene fusion clonings and the report of expression, but rarely found has It closes a variety of small peptide Gene Fusions for having antibacterial activity, and forms after coexpression multiple bioactive molecules and the anti-sense of Synergistic antimicrobial Dye improves the report of immune level.The present invention is merged by genetic recombination, and building new have antibacterium, virus, fungi and post The fusion antibacterial peptide gene (PR-39/Tritrpticin/PAMP23/PG1cDNA) of the various actives such as infested and immunological regulation, It is connected to eukaryon Yeast expression technology and obtains fusion protein, further pass through the immune of opposite MIC methods research fusion protein Activity and its antibacterial, antiviral activity are adjusted, to obtain the recombination egg that broad-spectrum high efficacy is antimicrobial and immunoregulation effect is strong In vain.

Invention content

The technical problem to be solved by the present invention is to how improve the immunocompetence of animal.

In order to solve the above technical problems, the present invention provides a kind of protein, entitled FPAP, including 4 boar antibacterial peptides；

The amino acid sequence of the 4 boar antibacterial peptide is respectively sequence 1 1-42, sequence 1 51-63, sequence 1 72-94 and sequence 1 103-129.

Above-mentioned protein be following a)-e) in any protein：

A) amino acid sequence includes the protein of amino acid sequence shown in sequence 1 in sequence table；

B) amino acid sequence amino acid residue shown in sequence in sequence table 1 forms；

C) by substitution and/or missing of the amino acid sequence by one or several amino acid residues defined by a) or b) And/or it adds and there is the protein for improving animal immune ability function；

D) amino acid sequence defined by and a) or b) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology of person and the protein with raising animal immune ability function；

E) a)-d) in it is any defined by protein N-terminal and/or C-terminal connection label after obtained fusion protein.

The nucleic acid molecules for encoding above-mentioned albumen are also the scope of protection of the invention.

Above-mentioned nucleic acid molecules are following 1) -4) in it is any shown in nucleic acid molecules：

1) its coded sequence includes sequence 2 in sequence table；

2) its coded sequence is sequence 2 in sequence table；

1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and encode the DNA molecular of above-mentioned albumen；

1) or 2) 4) with the DNA molecular that limits with 80% or more or 90% or more homology and the above-mentioned albumen of coding DNA molecular.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.

Wherein, FPAP shown in 2 coded sequence 1 of sequence.

Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side Method is mutated the nucleotide sequence of the coding FPAP of the present invention.Those have and are detached with the present invention by manually modified The nucleotide sequence 75% of obtained FPAP or the nucleotide of higher homogeneity, as long as encoding FPAP and having the function of FPAP, It is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.

Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair Shown in bright coded sequence 1 amino acid sequence form protein nucleotide sequence have 75% higher or 85% or Higher or 90% higher or 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or computer software It is evaluated.Using computer software, homogeneity between two or more sequences can use percentage (%) to indicate, can be with For evaluating the homogeneity between correlated series.

In above application, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times, each 5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min； Or, in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS, hybridizes under the conditions of 65 DEG C and wash film.

Above-mentioned 75% or 75% or more homogeneity can be 80%, 85%, 90% or 95% or more homogeneity.

Following 1) -3) any one of biomaterial is also the scope of protection of the invention：

1) contain the expression cassette of above-mentioned nucleic acid molecules；

2) contain the recombinant vector of above-mentioned nucleic acid molecules；

3) contain the recombinant bacterium or transgenic cell line of above-mentioned nucleic acid molecules；

4) tunning of the recombinant bacterium.

In above application, B2) described in the nucleic acid molecules containing coding FPAP expression cassette (FPAP expression casettes), be Refer to express the DNA of FPAP in host cell, which not only may include the promoter for starting FPAP genetic transcriptions, may be used also Terminator including terminating FPAP genetic transcriptions.Further, the expression cassette may also include enhancer sequence.

The recombinant vector of the FPAP expression casettes can be contained with existing vector construction.

In above application, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.The plasmid is concretely PGAPZ α A carriers.

B3) recombinant vector contains the DNA sequence dna for encoding FPAP shown in sequence 2.Recombination described further Carrier concretely pG-p.The pG-p is that EcoR I and the Xba I of pGAPZ α A carriers are identified that the DNA fragmentation between sequence replaces It is changed to FPAP genes shown in sequence 2, obtained recombinant vector.

In above application, the microorganism can be yeast, bacterium, algae or fungi.Wherein, yeast can be Pichia pastoris SMD1168。

In above application, the recombinant microorganism can be that the expression cassette for the nucleic acid molecules that will contain coding FPAP imports yeast In obtained recombinant microorganism.The recombinant microorganism will concretely be imported containing the recombinant vector of FPAP expression cassettes in yeast Obtained recombinant microorganism.Recombinant vector is being imported in the yeast, can directly imported recombinant vector in the yeast, It can show and be imported again in the yeast after linearizing recombinant vector.The yeast can be Pichia pastoris SMD1168.

In one embodiment of the invention, the recombinant microorganism is that the pG-p is imported Pichia pastoris SMD1168 In obtained recombinant microorganism.The preparation method of the recombinant microorganism specifically includes：The pG-P is linearized, is obtained linear Change pG-P；The linearisation pG-P is imported in Pichia pastoris SMD1168, obtains recombinant microorganism, the name of the recombinant microorganism Referred to as SMDpG-p.

In above application, the tunning of the recombinant microorganism can be prepared according to the method included the following steps：Culture The recombinant microorganism makes the encoding gene of FPAP express, obtains the tunning of the recombinant microorganism.

In above application, the transgenic cell line does not include propagating materials.

The application of above-mentioned protein or above-mentioned nucleic acid molecules or above-mentioned biomaterial in following C1 or C2 is also this hair The range of bright protection：

C1, animal immune ability is being improved；

C2, it prepares and improves animal immune ability product.

In above application, the raising animal immune ability is at least one of following M1-M5：

M1, the growth for inhibiting pathogenic microorganisms；

M2, the increase for promoting immunocyte；

M3, promote vaccine-induced immune response；

M4, promote cellular immunity and/or humoral immunity；

M5, animal development and growth-weight gain are improved；

And/or the pathogenic microorganisms is specially Escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, pig breeding With disordered breathing syndrome virus or swine fever virus, such as Escherichia coli standard bacteria (G^-), Escherichia coli drug-fast bacteria (G^-), it is golden yellow Staphylococcus standard bacteria (G⁺), staphylococcus aureus resistance bacterium (G⁺)。

And/or the immunocyte is specially lymphocyte (such as CD4+ or CD8+), red blood cell or leucocyte；

And/or the antibody specific is IgG, IgG1 and/or IgG2a.The antibody specific can be the pathogenic microorganisms Antibody.

Any products of following X1 or X2 are also the scope of protection of the invention：

X1, biological agent contain following X3a, X3b or X3c：

X3a, above-mentioned protein；

X3b, above-mentioned nucleic acid molecules；

X3c, above-mentioned biomaterial；

X2, the reagent set for improving animal immune ability, by above-mentioned X1 and antibiotic group at.

In the said goods, the biological agent can using X3a, X3b or X3c as active constituent, can also by X3a, X3b or X3c is active constituent with the composition that other substances that can improve animal immune ability are combined.

Or, a kind of method improving animal immune ability, including above-mentioned protein or above-mentioned nucleic acid molecules are applied to animal Or above-mentioned biomaterial or the biological agent or the reagent set, improve the immunocompetence of the animal.

Among the above, the animal is any one of H1-H3：

H1, mammal；

H2, pig；

H3, mouse.

In the said goods, the antibiotic can be ammonia benzyl mycin and/or kanamycins.

In order to solve the above technical problems, the present invention also provides the method for improving animal immune ability, the method can wrap It includes and FPAP, the biomaterial or the biological agent is applied to animal, improve the immunocompetence of the animal.

In the present invention, the raising animal immune ability can be any one of following M1-M5：

M1, the growth for inhibiting pathogenic microorganisms；

M2, the increase for promoting immunocyte；

M3, promote vaccine-induced immune response；

M4, promote cellular immunity and/or humoral immunity；

M5, animal development and growth-weight gain are improved.

The pathogenic microorganisms can be Escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, pig breeding and breathing barrier Hinder syndrome virus (PRRSV) or swine fever virus (HCV or CSFV), such as Escherichia coli standard bacteria (G^-), Escherichia coli drug-fast bacteria (G^-), staphylococcus aureus standard bacteria (G⁺), staphylococcus aureus resistance bacterium (G⁺)。

The immunocyte can be lymphocyte (such as CD4+ or CD8+), red blood cell or leucocyte.

The antibody can be IgG, IgG1 and/or IgG2a.The antibody specific can be the antibody of the pathogenic microorganisms.

The vaccine is concretely directed to the vaccine of the pathogenic microorganisms.

In the present invention, the raising animal immune ability concretely improves the animal and exempts to the pathogenic microorganisms Epidemic disease ability such as improves the immunocompetence of the mycoplasma hyopneumoniae to causing swine enzootic pneumonia (MP).

In the present invention, the animal can be mammal；The mammal concretely pig or mouse.

In the present invention, the raising animal immune ability product can be the drug for improving animal immune ability.

It is demonstrated experimentally that the coexpression FPAP molecules of the present invention and the tunning of the recombination yeast containing FPAP genes have It acts below：The increase for promoting animal lymph cell, red blood cell and leucocyte, using the animal body endolymph cell of FPAP, red Cell and leucocyte content can be improved 10% or more；The apparent growth for inhibiting pathogenic microorganisms, using the pathogenic microorganisms of FPAP Amount can reduce up to 20% or more；The secretion for promoting non-specific antibody (IgG, IgG1, IgG2a) and disease specific antibody, is applied 40%-60% can be improved with the content of non-specific antibody and disease specific antibody in the animal body of FPAP；Promote immune correlation The expression of gene, and then the immune anti-infection ability of animal is improved, it attacks malicious survival rate using the animal of FPAP and significantly improves at least 60%, 10% or more weight gain.

Description of the drawings

Fig. 1 is the RT-PCR results of target gene in SMDpG-P.

Fig. 2 be SMDpG-P in target gene protein expression level testing result.

Fig. 3 is the influence that yeast strain SMDpG-P fermented supernatant fluids are proliferated pig lymphoblast.

Fig. 4 is inhibiting effect of the SGP to staphylococcus aureus standard bacteria.

Fig. 5 is inhibiting effect of the SGP to staphylococcus aureus resistance bacterium.

Fig. 6 is inhibiting effect of the SGP to Escherichia coli standard bacteria.

Fig. 7 is inhibiting effect of the SGP to Escherichia coli drug-fast bacteria.

Fig. 8 is that mouse peripheral blood leucocyte changes with time under different processing.

Fig. 9 is the variation of CD4+T lymphocytes in every 10000 cells of mouse peripheral blood.

Figure 10 is the variation of CD8+T lymphocytes in every 10000 cells of mouse peripheral blood.

Figure 11 is the variation of different group mouse peripheral blood IgG levels.

Figure 12 is the variation of different group mouse peripheral blood IgG1 levels.

Figure 13 is the variation of different group mouse peripheral blood IgG2a levels.

Figure 14 is different group mouse peripheral blood MP specific antibody contents.

Figure 15 is different group mouse peripheral blood TNF-α gene expression doses.

Figure 16 is different group mouse peripheral blood IL-4 gene expression doses.

Figure 17 is different group mouse peripheral blood TLR gene expression doses.

Figure 18 is different group mouse peripheral blood immunological memory correlation factor gene expression doses.

Figure 19 is different group mouse survival rate after Escherichia coli and staphylococcus aureus attack poison.

Figure 20 is that each group piglet has a net increase of weight.

The dynamic change of quantity of leucocyte during Figure 21 is piglet experiment.

Figure 22 is the dynamic change of CD8+T lymphocytes in peripheral blood during piglet is tested.

Figure 23 is the dynamic change of CSF specific antibodies in peripheral blood during piglet is tested.

Figure 24 is the dynamic change of PRRSV specific antibodies in peripheral blood during piglet is tested.

Peripheral blood TLR gene expression doses during Figure 25 is piglet experiment.

Peripheral blood immune factor gene expression dose during Figure 26 is piglet experiment.

Specific implementation mode

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

PGAPZ α A carriers in following embodiments are Invitrogen Products, catalog number V20020.

Pichia pastoris SMD1168 in following embodiments is Invitrogen Products, catalog number C17500.

Landrace in following embodiments is Sichuan Province boar performance measurement center Jianyang base product.

Tibetan pig in following embodiments is Sichuan Province boar performance measurement center Jianyang base product.

Escherichia coli standard bacteria (G in following embodiments^-) it is ATCC (America Type Culture Collection) product, catalog number 25922.

Escherichia coli drug-fast bacteria (G in following embodiments^-) it is Sichuan University's Field of Animal Epidemic Disease Control and food security Sichuan Key lab of province provides, and the public can obtain the biomaterial from applicant, which only attaches most importance to the phase of duplicate invention It closes used in experiment, not can be used as other purposes and use.

Staphylococcus aureus standard bacteria (G in following embodiments⁺) it is ATCC (America Type Culture Collection) product, catalog number 29213.

Staphylococcus aureus resistance bacterium (G in following embodiments⁺) it is that Sichuan University's Field of Animal Epidemic Disease Control is pacified with food Full Key Laboratory of Sichuan Province provides, and the public can obtain the biomaterial from applicant, which only attaches most importance to duplicate hair Used in bright related experiment, it not can be used as other purposes and use.

Female ICR mice in following embodiments is People's Hospital, Sichuan Prov.'s institute of lab animals's product.

Embodiment 1, pig antibacterial peptide fusion protein (FPAP) and its encoding gene

One, the acquisition of fusion protein F PAP and its encoding gene

By 4 boar antibacterial peptides by connecting peptide fusion, fusion protein, entitled FPAP are obtained.

In the amino acid sequence of the fusion protein such as sequence table shown in sequence 1, the fusion base of fusion protein F PAP is encoded Because being named as FPAP, the nucleotides sequence of the fusion is classified as sequence 2.

Wherein, sequence 1 1-42 is pig antibacterial peptide PR-39, and 43-50 are connection peptide, and 51-63 are pig antibacterial Peptide Tritrpticin, 64-71 are connection peptide, and 72-94 are pig antibacterial peptide PAMP23, and 95-102 are connection peptide, 103-129 are pig antibacterial peptide PG1.

Sequence 2 1-126 is pig antibacterial peptide PR-39 code nucleic acid, and 127-150 are connection peptide code nucleic acid, the 151-189 are pig antibacterial peptide Tritrpticin code nucleic acids, and 190-213 are connection peptide code nucleic acid, 214-282 Position is pig antibacterial peptide PAMP23 code nucleic acids, and 283-306 are connection peptide code nucleic acid, and 307-387 are pig antibacterial peptide PG1 code nucleic acids.

Above-mentioned albumen can be by artificial synthesized code nucleic acid, and prokaryotic expression obtains.

Two, the preparation of expressed fusion protein FPAP recombinant vectors

Recombinant vector pGAPZ α A-B (abbreviation pG-p) are between EcoR I and Xba the I identification sequences by pGAPZ α A carriers DNA fragmentation replace with FPAP genes shown in sequence 2, keep the other sequences of carrier constant, obtain recombinant vector, the carrier Pig antibacterial peptide fusion protein (FPAP) shown in expressed sequence 1.

Three, prepared by the recombinant bacterium of expressed fusion protein FPAP

1, prepared by recombinant bacterium

With AvrII digestion pG-p, linearisation pG-p is obtained；Linearisation pG-p is imported in Pichia pastoris SMD1168, is obtained The recombination yeast is named as SMDpG-p by recombination yeast.

Linearisation pGAPZ α A are obtained with AvrII digestion pGAPZ α A using same method；It will linearisation pGAPZ α A is imported in Pichia pastoris SMD1168, obtains control recombinant bacterium SMDpG.

2, recombinant bacterium detects

1) detection of expression on rna level

Recombinant bacterium SMDpG-p is seeded in the YPD culture mediums containing 100 μ g/ml Zeocin 28 DEG C, 200rpm is overnight Culture, centrifugation, obtains SMDpG-p thalline.SMDpG-p thalline total serum IgEs are extracted, with primer pair (F：5’- ATGTATAAGATGCAGCTCTTGT-3 ' and R：5'-CTAATGGTGATGGTGATGAT-3') detect target gene in SMDpG-p Expression quantity.

The results are shown in Figure 1, Lane M:20bp DNA Ladder Marker；Lane 1-2:The RT-PCR of SMDpG-P Amplified band；There are 405bp purpose bands in SMDpG-p, shows that the fusion FPAP in SMDpG-p has obtained table in rna level It reaches.

Control recombinant bacterium SMDpG is detected using same method, without the expression of fusion FPAP.

2), protein expression level detects

Recombinant bacterium SMDpG-p (SGP) is seeded in the YPD culture mediums containing 100 μ g/ml Zeocin 28 DEG C, 200rpm Fermented and cultured takes zymotic fluid to be centrifuged, supernatant is taken, with the ELISA kit of HIS-Tag in fermentation different time respectively (all antibody test reagents are all inside) detects the fusion protein in supernatant.With control recombinant yeast pichia pastoris SMD1168 The obtained supernatant that ferments (compares recombinant bacterium SMDpG as control, result has been displayed in fig. 2, OD as negative control₄₅₀ <0.02, it is far below SGP fermented supernatant fluids).

The results are shown in Figure 2, and compared with Pichia pastoris SMD1168, recombinant bacterium SMDpG-p (SGP) generates purpose and merges egg White FPAP, and in the expression quantity highest of fermentation 72 hours.

The above results show recombinant bacterium SMDpG-p expression purpose fusion protein Fs PAP.

Embodiment 2, pig antibacterial peptide fusion protein (FPAP) are on lymphopoietic influence

1, the preparation of recombinant bacterium SMDpG-p fermented supernatant fluids

(1) the recombinant bacterium SMDpG-p (hereinafter referred to as SGP) that embodiment 1 obtains is inoculated in (the culture medium 1 of 3mL culture mediums 1 For the fluid nutrient medium that bleomycin (Zeocin) obtains, and a concentration of 100mg/ of bleomycin are added into YPD culture mediums ML in), 28 DEG C, 200rpm overnight incubation activated spawns.

(2) bacterium solution that 300 μ L steps (1) obtain is taken to be inoculated in the 100mL triangular flasks of the culture mediums of YPD containing 30mL, 28 DEG C, 200rpm shaker fermentation culture 48h (OD₆₀₀For 25).

(3) bacterium solution for taking 5mL steps (2) to obtain, 2min is centrifuged under 12 000 × g, and obtained supernatant is named as SGP fermented supernatant fluids.

2, Protease Treatment recombinant bacterium SMDpG-p fermented supernatant fluids

The pH for the SGP fermented supernatant fluids that above-mentioned 1 obtains first is surveyed with pH test paper, then with 2mol/L NaOH and 1mo/L HCl points Not Tiao SGP fermented supernatant fluids to the most suitable action pH 7.0 of trypsase and the most suitable action pH 2.0 of pepsin, respectively plus Enter trypsin solution (Beijing Suo Laibao Science and Technology Ltd, trypsase-EDTA digestive juices (Trypsin-EDTA Solution) contain 0.25% pancreatin and 0.02%EDTA, catalog number (Cat.No.)：9002-07-7) and pepsin solution (Beijing Suo Laibao Science and Technology Ltd., 0.1% solution ph 4.0, enzyme activity:3000-3500NFU/g, catalog number (Cat.No.)：9001-75-6) simulation digestion The effect of road enzyme protein degradation, enables fusion protein independently to separate, and protease hydrolytic falls to link sequence " GSGDDDDK ", makes enzyme Final concentration is 0.5mg/mL, and 37 DEG C of water-bath enzyme reaction 1h obtain the SGP fermented supernatant fluids and pepsin of trypsin treatment The SGP fermented supernatant fluids of processing, it is spare in -20 DEG C of refrigerators.

According to the method described above, SMDpG-p is replaced with into SMDpG (hereinafter referred to as SG), other steps are constant, respectively obtain The SG fermented supernatant fluids of SG fermented supernatant fluids, the SG fermented supernatant fluids of trypsin treatment and pepsin, in -20 DEG C of ice Case is spare.

3, fusion protein F PAP is on lymphopoietic influence

1), the preparation of Landrace lymphocyte

Aseptically, Landrace vena cava anterior peripheral blood 5mL is taken with heparin tube (anti-freezing containing EDTA-2K), according to Pig lymphocyte separating liquid (using first 37 DEG C preheating separating liquids and fully shaking mixing) operating procedure carries out separation Landrace and drenches Bar cell.

2), SMDpG-p tunnings In vitro biological activity detects

(1) by after the pig lymphocyte culture for 24 hours of above-mentioned 1) separation, the Landrace lymphoblast in culture dish is shifted Into clean 15mL sterile centrifugation tubes, 1500rpm room temperatures centrifuge 15min and collect cell body.

(2) cell is washed with RPMI1640 complete mediums (dual anti-, 10% fetal calf serum containing mycillin), 2 times repeatedly, 1500rpm room temperatures centrifuge 15min and collect cell precipitation.

(3) with the RPMI1640 complete medium gravity treatment cells of-MM of α containing 20mg/mL and adjust number of cells be about 6 × 10⁶A/mL, obtains cell suspending liquid.

(4) according to typesetting to 96 porocyte plates per be added in hole the cell suspending liquids of 75 μ L steps (3), 45 μ L samples liquid and The RPMI1640 complete mediums of 30 μ L α containing 20mg/mL-MM (methyl mannoside).

Wherein sample liquid be respectively step 1 and 2 obtain SGP fermented supernatant fluids, trypsin treatment SGP fermentation supernatants Liquid, the SGP fermented supernatant fluids of pepsin, SG fermented supernatant fluids, the SG fermented supernatant fluids of trypsin treatment and stomach egg The SG fermented supernatant fluids of white enzymatic treatment；Per a kind of sample liquid in hole, three multiple holes of each sample liquid.

The cell that the RPMI1640 complete mediums for containing only 20mg/mL α-MM, PBS and step (3) is respectively adopted suspends Blank control is used as in liquid.

It is positioned over 5%CO₂, 37 DEG C of cell incubator culture 48h.

(5) 96 porocyte plates are taken out, 15 μ L CCK8 (the Guangzhou bio tech ltd Yi Yuan) gently mixings are added per hole After be put into 5%CO₂, 37 DEG C of cell incubators continue to cultivate 2h, 96 porocyte plates are taken out, with microplate reader (Bio-Reader3350) Detection is per hole OD₄₅₀。

The results are shown in Figure 3 (blank control is used as in the cell suspending liquid of step (3)), it can be seen that regardless of whether through Trypsase or pepsin are crossed, compared with control group recombinant bacterium SG fermented supernatant fluids, SGP fermented supernatant fluids stimulate pig The lymphocyte that lymphoblast obtains dramatically increases (P<0.05)；Show that fusion protein F PAP significantly can stimulate pig to drench Bar cell Proliferation (P<0.05).

In Fig. 3, fermented supernatant fluid of the untreated expression without trypsin treatment and without pepsin.

The bacteriostatic activity detection of embodiment 3, pig antibacterial peptide fusion protein (FPAP)

Pig antibacterial peptide fusion protein FPAP is measured to Escherichia coli standard bacteria (G^-) (hereinafter referred to as S-G^-), Escherichia coli Drug-fast bacteria (G^-) (hereinafter referred to as R-G^-), staphylococcus aureus standard bacteria (G⁺) (hereinafter referred to as S-G⁺), golden yellow grape Coccus drug-fast bacteria (G⁺) (hereinafter referred to as R-G⁺) antibacterial situation, the specific method is as follows：

4 kinds of bacterium bacterial strain inoculations are activated and cultivate it first and are in exponential phase of growth (OD₆₀₀About 0.5), it then uses LB culture solutions are diluted to OD₆₀₀About 0.005, the bacterium solution after dilution is inoculated into 96 porocyte culture plates, 100 μ L/ are per hole, often A kind of a bacterium of 96 porocyte culture plates.

It is handled in the following way containing germy 96 porocyte culture plates for each：100 μ L sample liquid are added real Gently mixing, each sample set 3 repeating holes in verifying；

Wherein sample liquid is the step 1 of embodiment 2 and the SGP fermented supernatant fluids of 2 acquisitions (in i.e. untreated SGP fermentations Clear liquid), the SGP fermented supernatant fluids of the SGP fermented supernatant fluids of trypsin treatment, pepsin, and by the step of embodiment 2 Rapid 1 and 2 SG fermented supernatant fluids, the SG fermented supernatant fluids of trypsin treatment and the SG fermentation supernatants of pepsin obtained Liquid is as corresponding empty map.

Each bacterium is all provided with four kinds of antibiotic gradients as positive control (antibiotic and LB culture solutions)；Each bacterium, which is all provided with, only to be added LB culture solutions set not bacteria-containing LB culture solutions as blank control as negative control.

After 96 porocyte culture plates are set 37 DEG C of incubators incubations 2h, 16h, microplate reader (Bio-Reader3350) is used respectively Detection is per hole OD₆₀₀Value.

S-G^-And S-G⁺Four kinds of antibiotic gradients it is as shown in table 1, R-G^-And R-G⁺Four kinds of antibiotic gradients such as table Shown in 2.

Table 1, reference culture antibiotic gradient

Note：In table 1, " --- " indicates to be free of kanamycins.

Table 2, antibody-resistant bacterium antibiotic gradient

The results show that for each bacterium in these four bacterium, the OD of the various sample liquids of SG₆₀₀With negative control OD₆₀₀In 16h without significant difference, show that SG acts on the equal unrestraint of these four bacterium；The OD of the various sample liquids of SGP₆₀₀ The OD of the empty map under the corresponding time is substantially less than in 16h₆₀₀(P<0.05)。

As shown in Figure 4-Figure 7 (empty map is SG fermented supernatant fluids), the fermented supernatant fluid of SGP is to this for result when 16h Four kinds of bacterium have apparent inhibiting effect.Show that fusion protein F PAP can inhibit Escherichia coli standard bacteria, Escherichia coli drug resistance Bacterium, staphylococcus aureus standard bacteria and staphylococcus aureus resistance bacterium growing multiplication.

Embodiment 4, pig antibacterial peptide fusion protein (FPAP) biological activity research in Mice Body

1, the preparation of tunning

Fermentation：The recombinant bacterium SMDpG-p (hereinafter referred to as SGP) that embodiment 1 obtains is activated and is inoculated in YPD containing 30mL In the 100mL triangular flasks of culture medium, 48h is cultivated under 30 DEG C, 220rpm, makes OD₆₀₀About 25, obtain SGP zymotic fluids.

According to the method described above, by control recombinant bacterium SMDpG fermentations, SG zymotic fluids are obtained.

2, ICR mice groups are tested

50 3 week old Healthy female ICR mouse of 18-20g are taken, random 5 groups of grouping, every group of 10 mouse, group, which is numbered, is 1-5, wherein group 1, group 4 are SG negative control groups, group 3 is vaccine negative control group, and group 2, group 5 are experimental group.

3, Mouse feeder and vaccine inoculation

According to grouping situation, fresh fermentation broth is sent to Mouse Stomach with gastric perfusion needle, only, first time gavage is remembered by 0.6mL/ For gavage the 0th day, gavage was primary every three days, 4 weeks continuous (i.e. respectively gavage the 0th day, the 3rd day, the 6th day, the 9th day, the 12nd It, the 15th day, the 18th day, the 21st day, the 24th day and the 27th day corresponding zymotic fluid of gavage, each gavage amount is 0.6mL/ Only).To ensure recombinant protein activity, fresh recombinant yeast pichia pastoris bacterium solution fermentation is carried out before each animal gavage.

When vaccine inoculation, the only intramuscular injection (intramuscular injection) in gavage the 7th day, 0.2mL/ is only.

Concrete operations are (table 3) as follows, wherein swine enzootic pneumonia (MP) vaccine is the limited public affairs of magnificent growth engineering group Take charge of the product (catalog number (Cat.No.) of production：19200003).

Table 3, intragastric administration on mice, vaccination doses

In table 3, gavage is the vaccination ways of zymotic fluid, and 0.6mL/ is only the dosage of inoculation of zymotic fluid；Intramuscular injection is The vaccination ways of vaccine, 0.2mL/ are only the dosage of inoculation of vaccine.

Respectively before gavage, gavage the 7th day, gavage the 14th day, gavage the 21st day and gavage take every mouse within the 28th day Tail vein carries out following experiment content：After 30 μ L whole bloods and 30 μ L physiological saline mixings blood routine is done in blood counting instrument；50 μ L whole bloods do Flow cytometry；100 μ L whole bloods extract real-time quantitative gene involved in immunity after RNA；100 μ L whole bloods add 1mL - 80 DEG C of the violent mixings of TRIZOL save backup；200 μ L whole blood low-speed centrifugals collect blood plasma and detect antibody.

4, challenge viral dosage

By the lethal Escherichia coli of high drug resistance, (Sichuan University's Field of Animal Epidemic Disease Control is carried with food security Key Laboratory of Sichuan Province For catalog number (Cat.No.)：SCSU-ECOLI-HRL-1012) it is inoculated in the benzyl mycin of ammonia containing 0.1mg/ml and 0.1mg/ml kanamycins LB liquid It is activated in body culture medium, the fresh bacterium solution after activation is inoculated in LB liquid medium 37 DEG C, 1500rpm is cultivated to logarithmic phase Thalline were collected by centrifugation, is resuspended to 5.0 × 10 with fresh LB liquid medium⁵CFU/ml obtains escherichia coli fermented broth. The half lethal dose that preliminary experiment gropes 7 week old Healthy female ICR mouse of escherichia coli fermented broth pair is 0.1ml.Gavage the 28th day 5 mouse peritoneal Escherichia Coli Injection zymotic fluids are randomly selected in each group, the injection volume of every mouse is half lethal dose, The intraperitoneal injection same day is to attack after poison the 0th day.Per the incidence of mouse of observation for 24 hours, and mouse survival rate is counted, dissected Observe the variation of dead mouse internal.

By the lethal staphylococcus aureus of high drug resistance, (Sichuan University's Field of Animal Epidemic Disease Control is real with food security Sichuan Province emphasis Room offer, catalog number (Cat.No.) are provided：SCSU-STREPC-HRL-2026) be inoculated in the benzyl mycin of ammonia containing 0.1mg/ml and 0.1mg/ml cards that It is activated in mycin LB liquid medium, the fresh bacterium solution after activation is inoculated in LB liquid medium 37 DEG C, 1500rpm cultures To logarithmic phase, thalline were collected by centrifugation, is resuspended to 5.0 × 10 with fresh LB liquid medium⁵CFU/ml obtains golden yellow Staphylococcal fermentation liquid.Preliminary experiment gropes the semilethal agent of 7 week old Healthy female ICR mouse of S. aureus fermentation liquid pair Amount is 0.2ml.Gavage the 28th day injects 5 mouse peritoneals of remaining non-Escherichia Coli Injection zymotic fluid in every group golden yellow The injection volume of Staphylococcal fermentation liquid, every mouse is half lethal dose, and the intraperitoneal injection same day is also to attack after poison the 0th day.Often The incidence of a mouse is observed for 24 hours, and counts mouse survival rate, and the variation of dead mouse internal is dissected and observed.

5, analysis of experimental results

The results are shown in Figure 8 for every group of mouse blood routine detection peripheral white blood cells variation, and SG indicates blank control bacterium hair The content of zymotic fluid, experimental mice peripheral white blood cells is significantly higher than SG negative control groups and vaccine negative control group (P< 0.05)；SMDpG-p tunnings can effectively stimulate immune cell propagation, show fusion protein F PAP effectively and stimulate and is immune Cell Proliferation.

Sample is handled with reference to fluidic cell operating procedure, is protected from light machine testing Th on low temperature (CD4+ lymphocytes), Tc (CD8+ Lymphocyte) cell quantity, Flow cytometry result such as Fig. 9 and Figure 10 show and randomly select every 10000 of mouse peripheral blood The variation of CD4+ and CD8+T lymphocytes in cell；As seen from the figure, the experiment mice peripheral blood CD4+ after immunity inoculation and The content of CD8+ is all remarkably higher than SG negative control groups and vaccine negative control group (P<0.05) it, is all reached within 21 or 28 days after immune To peak value.Illustrate that SMDpG-p tunnings have the function of stimulation test mouse immune response, shows that fusion protein F PAP is effective Ground stimulates immune cell propagation.

The blood plasma that low-speed centrifugal is collected according to ELISA kit operating procedure detection non-specific antibody IgG, IgG1, IgG2a, and the antibody titer of specific antibody MP antibody is detected, the kit of use is respectively mouse immune globulin G1 (IgG1) ELISA kit (article No. 69-210245), mouse immune globulin G (IgG) ELISA kit (article No. 59- 20037), mouse immune globulin G (IgG2a) ELISA kit (article No. 69-210250), porcine mycoplasmal antibody elisa reagents Box (article No. 69-40349), above each kit are the silent picogram bio tech ltd product in Wuhan.

As a result as shown in Figure 11,12,13, show IgG, IgG1 in experiment mice peripheral blood serum after immunity inoculation, IgG2a levels dramatically increase (P compared with SG negative control groups and vaccine negative control group<0.05) it, is all reached within 14 or 28 days after immune To peak value.Illustrate that SMDpG-p tunnings can stimulate and generates more IgG, IgG1, IgG2a antibody in immune Mice Body.Figure 14 Show the antibody titer of MP (mycoplasma) in the experiment mice peripheral blood serum after immunity inoculation, as time increases, Each group of antibody titer is gradually reduced, but experimental group is significantly higher than SG negative control groups and vaccine negative control group (P< 0.05).It illustrates that SMDpG-p tunnings can significantly improve vaccine-induced immune response effect, shows fusion protein FPAP, which is effectively stimulated, generates antibody.

Expression of the mouse immune related gene on rna level is detected, RNA is extracted as template, with table 4 using whole blood Shown in primer expanded, detect mouse immune related gene expression, reference gene be actin β-actin.

Table 4 is primer

Gene	Primer	Primer sequence (5 ' -3 ')
			TLR-1	TLR-1-F	GGACCTACCCTTGCAAACAA
	TLR-1-R	GGTGGCACAAGATCACCTTT
			TLR-4	TLR-4-F	ATATGGCAGAGGTGAAAGCAC
	TLR-4-R	GAAGGCAGAGATGAAAAGGGG
			TNF-α	TNF-α-F	CCTGTAGCCCACGTCGTAG
	TNF-α-R	GGGAGTAGACAAGGTACAACCC
			IL-4	IL-4-F	GCCATATCCACGGATGCGACAA
	IL-4-R	GGTGTTCTTCGTTGCTGTGAGGA
			IL-7	IL-7-F	TTCCTCCACTGATCCTTGTTCT
	IL-7-R	AGCAGCTTCCTTTGTATCATCAC
			IL-23	IL-23-F	TCAGACATTTTCACAGGGAGC
	IL-23-R	ACCCTCGGGCTGCAAGAGT

TNF-α is important Th1 cytokines, is primarily involved in differentiation and the cellular immunity of Th1 cells, dynamic change As shown in figure 15, experimental group is significantly higher than SG negative control groups and vaccine negative control group (P<0.05), and after immunity inoculation Reach peak value within 21st day.Th2 cells secrete IL4 cell factors, participate in the humoral immunity of body, dynamic change such as Figure 16 institutes Show, experimental group is significantly higher than SG negative control groups and vaccine negative control group (P<0.05), and at 14 to 21 days reach peak value.Knot Fruit shows that SMDpG-p tunnings can promote cellular immunity and humoral immunity, i.e. fusion protein F PAP that can promote simultaneously simultaneously Into cellular immunity and humoral immunity.

Figure 17 is the dynamic change of TLR gene expression doses, after as a result showing immunity inoculation, TLR1 genes and TLR4 genes Expression obviously rise (P<0.05), and the TLR gene expression doses of experimental mice are compared with SG negative control groups and vaccine Negative control group dramatically increases (P<0.05), reach peak value within 21 days after immune.

Figure 18 is the dynamic change of immunological memory related gene expression level, overall to show, after immunity inoculation, experimental group is small The IL-23 genes and IL-7 gene expression doses of mouse dramatically increase (P compared with SG negative control groups and vaccine negative control group< 0.05)。

It attacks after poison the 5th day, for the mouse of intraperitoneal injection escherichia coli fermented broth, the mouse survival rate of experimental group is significantly high In SG negative control groups and vaccine negative control group (Figure 19), for the mouse of intraperitoneal injection S. aureus fermentation liquid, The mouse survival rate of experimental group is significantly higher than SG negative control groups and vaccine negative control group, illustrates the SMDp46B energy of experimental group It is enough effectively protected mouse, so that it is significantly increased the resistance of Escherichia coli and staphylococcus aureus, i.e. fusion protein FPAP can improve mouse and be attacked the survival rate after poison by pathogenic bacteria.Dead mouse is found by dissection, Escherichia coli are lethal Mouse peritoneal digested road has apparent lesion, spleen to black, and liver blacks, the lethal mouse peritoneal of infection of staphylococcus aureus Digested road blacks without apparent lesion, spleen, and liver is normal.

Embodiment 5, fusion protein F PAP are studied in piglet Biological acdtivity in vivo

1, the preparation of tunning

Fermentation：The recombinant bacterium SMDpG-p (hereinafter referred to as SGP) that embodiment 1 obtains is activated and is inoculated in YPD containing 30mL In the 100mL triangular flasks of culture medium, 48h is cultivated under 30 DEG C, 220rpm, makes OD₆₀₀About 40, obtain SGP zymotic fluids.

According to the method described above, by control recombinant bacterium SMDpG fermentations, SG zymotic fluids are obtained.

2, experimental animal feeds zymotic fluid

45 ages in days Du for choosing 18 weight about 9kg greatly enhances hybridization piglet, by Sichuan Province boar performance measurement center Jianyang Base provides, and is randomly divided into experimental group (9) and control group (9).To the SGP zymotic fluids of every pig feeding step 1 of experimental group, To the SG zymotic fluids of every pig feeding step 1 of control group, scale of feeding is 12.5ml/kg weight, is fed 28 days, is raised every 1 day It feeds once, feeding the previous day first time will be denoted as nursing 0 day.All experiment pigs receive identical swine fever attenuated vaccine (in herd Share Chengdu medical instruments factory, catalog number；And blue otopathy inactivated vaccine (Zhongmu Stocks Trading Co. Chengdu medical instruments factory, product 220051001) Catalog number (Cat.No.)；22003) general muscle injecting immune；It takes a blood sample respectively to all piglets nursing 0 day, 7 days, 14 days, 28 days, 42 days Vena cava anterior blood 3-4mL in containing EDTA-K2 vacuum tubes, be subsequently used for blood sample immunocyte variation, feed 0 day, 14, It weighs to all piglets within 28 days, 42 days.

3, experiment pig weight variation

It weighs to each group experiment piglet feeding 0 day, 14 days, 28 days and 42 days, as a result (Figure 20) is shown, experimental group Piglet is respectively the 1.71 of control group, 1.37 and 1.19 times feeding 14 days, 28 days and 42 days average increased weight, and difference is equal Reach the level of signifiance (P ＜ 0.05)；Illustrate that SGP effectively promotes piglet growth.

4, the dynamic change of piglet peripheral white blood cell amount is tested

It will be mixed by group in 0 day, 7 days, 14 days, the 28 days and 42 days blood sample taken of nursing, use Routine blood cell Analyzer measures the quantity of leucocyte in blood sample.As a result (Figure 21) is shown, feeding, 42 days SGP experimental group quantity of leucocyte are notable Higher than SG control groups (P<0.05).Illustrate that fusion protein F PAP can effectively increase the peripheral blood immuning cell number using object Amount, to which enhancing is immune.

5, the detection of piglet peripheral blood CD8+T lymphocyte subgroups is tested

It will be mixed by group in 7 days, 14 days, the 28 days and 42 days blood samples taken of nursing, be used for FCM analysis The quantity of CD8+T lymphocyte subgroups in peripheral blood, using Mouse Anti-Porcine CD8a-SPRD (Southern Biotech companies, article No. 4520-13) and 1 μ l Mouse Anti-Porcine CD3 ε-FITC (Southern Biotech public affairs Department, article No. 4510-02) it carries out, it is as follows：

(1) taking fresh 100 μ l of anti-freezing porkling venous blood, (quantity of leucocyte is about 10⁵-10⁷It is a), 60 μ l physiology salts are added Water；

(2) 2 μ l Mouse Anti-Porcine CD8a-SPRD (Southern Biotech companies, article No.s are drawn 4520-13) and 1 μ l Mouse Anti-Porcine CD3 ε-FITC (Southern Biotech companies, article No. 4510-02) Into 1.5mlEP pipes, mixing is incubated 20min；

(3) 0.2ml 10x erythrocyte cracked liquids are added in flow cytometer Special test tube, adds 1.8mlPBS, will incubate The blood cultivated is added in lysate, cracks 5min, waits for that haemocyte cracking is complete；

(4) 1500rpm centrifuges 5min, abandons supernatant.2mlPBS is added, blows and beats mixing, suspension cell；

(5) 1500rpm centrifuges 5min, abandons supernatant, about 150 μ l PBS is stayed gently to blow and beat mixing, washs 1-2 times altogether, washing The dosage of liquid should usually be at least 5 times of cell precipitation volume；

(6) to be detected with 150 μ l PBS piping and druming mixing cells.

As a result such as Figure 22 is shown, is being fed 7 days, 14 days, 28 days and 42 days, SGP experimental group CD8+T lymphocyte quantities are equal It is significantly higher than SG control groups (P<0.05).Illustrate that fusion protein F PAP can effectively stimulate the immune response function of piglet.

6, the ELISA detections of CSF and PRRSV specific antibodies：

(hog cholera antibody (CSF Ab) ELISA kit, pig are blue according to ELISA kit for the blood plasma that low-speed centrifugal is collected Otopathy poison ELISA kit) operating procedure detect specific antibody.

Figure 23 and Figure 24 is respectively swine fever (CSF) specific antibody and blue otopathy (PRRS) in piglet growth period peripheral blood The dynamic change figure of specific antibody, in figure, it is apparent that feeding 0 day, 14 days, 28 days and 42 days, two kinds in SGP experimental groups The quantity of specific antibody is all remarkably higher than SG control groups (P<0.05).Illustrate that fusion protein F PAP can significantly enhance vaccine The immune response of induction, to increase the protective rate of vaccine.

7, the expression variation of gene involved in immunity

Detect expression of the piglet immunological related gene on rna level, gene involved in immunity and primer such as 5 institute of table Show, internal reference is actin β-actin.

Table 5, quantitative primer

Figure 25 is the dynamic change of TLR gene expression doses, after as a result showing immunity inoculation, TLR-4 and TRL-7 genes Expression obviously rises (P<0.05), and TLR-4 the and TLR7 gene expression doses of experiment pig are notable compared with SG negative control groups Increase (P<0.05).

Figure 26 is the dynamic change of gene involved in immunity expression, after as a result showing immunity inoculation, the IL- of experiment pig 10, the gene expression dose of IFN-r, IL-12, IL-23 SGP at 7-42 days, 7-28 days and 7-42 days are apparently higher than SG groups (P< 0.05)；The expression of CD45 and CD62L genes also obviously rises (P compared with control group<0.05), reach within 7-14 days after immune Peak value.

After these are the result shows that fusion protein F PAP can improve experiment pig swine fever (CSF) and blue otopathy (PRRS) vaccine immunity The expression of related immune gene enhances its congenital immunity, specificity humoral and cellullar immunologic response reaction.

Sequence table

<110>Hai Jinzhuo Bioisystech Co., Ltd before Shenzhen, Sichuan University

<120>Merge the preparation and application of pig antibacterial peptide FPAP recombination yeast bacteria preparations

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 135

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 1

Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro

1 5 10 15

Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro

20 25 30

Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg Gly Ser Gly Asp Asp Asp

35 40 45

Asp Lys Val Arg Arg Phe Pro Trp Trp Trp Pro Phe Leu Arg Arg Gly

50 55 60

Ser Gly Asp Asp Asp Asp Lys Arg Ile Ile Asp Leu Leu Trp Arg Val

65 70 75 80

Arg Arg Pro Gln Lys Pro Lys Phe Val Thr Val Trp Val Arg Gly Ser

85 90 95

Gly Asp Asp Asp Asp Lys Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg

100 105 110

Arg Phe Cys Val Cys Val Gly Arg Gly Gly Ser Gly Asp Asp Asp Asp

115 120 125

Lys His His His His His His

130 135

<210> 2

<211> 405

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 2

aggagacgtc cccgaccccc atatttgcca aggccaaggc cacctccgtt tttcccacca 60

aggttgccac cacgtatccc accagggttc ccaccaaggt tcccaccacg gttccccgga 120

aaacggggat ccggagatga cgatgacaag gtacgacgtt tcccatggtg gtggcctttc 180

ttgcgacgtg gatccggaga tgacgatgac aagaggatta ttgacttgtt gtggagagta 240

cgtcggccac agaaacccaa atttgtgact gtatgggtca gaggatccgg agatgacgat 300

gacaagaggg gaggtcgcct gtgctattgt aggcgtaggt tctgcgtctg tgtcggacga 360

ggaggatccg gagatgacga tgacaagcat catcaccatc accat 405

Claims (10)

1. a kind of protein, including pig antibacterial peptide PR-39, pig antibacterial peptide Tritrpticin, pig antibacterial peptide PAMP23 and pig antibacterial Peptide PG1；

The amino acid of the pig antibacterial peptide PR-39, pig antibacterial peptide Tritrpticin, pig antibacterial peptide PAMP23 and pig antibacterial peptide PG1 Sequence is respectively sequence 1 1-42, sequence 1 51-63, sequence 1 72-94 and sequence 1 103-129.

2. protein according to claim 1, it is characterised in that：The protein be following a)-e) in any albumen Matter：

A) amino acid sequence includes the protein of amino acid sequence shown in sequence 1 in sequence table；

B) amino acid sequence amino acid residue shown in sequence in sequence table 1 forms；

C) by amino acid sequence defined by a) or b) by the substitution of one or several amino acid residues and/or missing and/or Addition and the protein with raising animal immune ability function；

D) amino acid sequence defined by and a) or b) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology and the protein with raising animal immune ability function；

E) a)-d) in it is any defined by protein N-terminal and/or C-terminal connection label after obtained fusion protein.

3. encoding the nucleic acid molecules of albumen described in claims 1 or 2.

4. nucleic acid molecules according to claim 3, it is characterised in that：The nucleic acid molecules are following 1) -4) in it is any Shown in nucleic acid molecules：

1) its coded sequence includes sequence 2 in sequence table；

2) its coded sequence is sequence 2 in sequence table；

1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and encode the DNA molecular of albumen described in claim 1；

1) or 2) 4) with the DNA molecular that limits with described in 80% or more or 90% or more homology and coding claim 1 The DNA molecular of albumen.

5. following 1) -4) any one of biomaterial：

1) expression cassette containing the nucleic acid molecules of claim 3 or 4；

2) recombinant vector containing the nucleic acid molecules of claim 3 or 4；

3) recombinant bacterium or transgenic cell line containing the nucleic acid molecules of claim 3 or 4；

4) tunning of the recombinant bacterium.

6. the biological material described in protein or claim 3 or 4 nucleic acid molecules or claim 5 described in claims 1 or 2 Expect the application in following C1 or C2：

C1, animal immune ability is being improved；

C2, it prepares and improves animal immune ability product.

7. applying according to claim 6, it is characterised in that：The raising animal immune ability be following M1-M5 in extremely Few one kind：

M1, the growth for inhibiting pathogenic microorganisms；

M2, the increase for promoting immunocyte；

M3, promote vaccine-induced immune response；

M4, promote cellular immunity and/or humoral immunity；

M5, animal development and growth-weight gain are improved；

And/or the pathogenic microorganisms is specially Escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, pig breeding and exhales Inhale disorders syndrome virus or swine fever virus；

And/or the immunocyte is specially lymphocyte, red blood cell or leucocyte；

And/or the antibody specific is IgG, IgG1 and/or IgG2a.

8. any products of following X1 or X2：

X1, biological agent contain following X3a, X3b or X3c：

Protein described in X3a, claims 1 or 2；

X3b, claim 3 or 4 nucleic acid molecules；

Biomaterial described in X3c, claim 5；

X2, the reagent set for improving animal immune ability, by above-mentioned X1 and antibiotic group at.

9. a kind of method improving animal immune ability, including protein or right described in claims 1 or 2 are applied to animal and wanted Ask biological agent described in biomaterial or the claim 8 described in 3 or 4 nucleic acid molecules or claim 5 or it is described at Reagent is covered, the immunocompetence of the animal is improved.

10. applied described according to claim 6 or 7 or claim 9 described in method, it is characterised in that：The animal is H1- Any one of H3：

H1, mammal；

H2, pig；

H3, mouse.