CN113201523A

CN113201523A - Gene engineering lyase for specifically killing streptococcus suis and medical application thereof

Info

Publication number: CN113201523A
Application number: CN202110531815.2A
Authority: CN
Inventors: 顾敬敏; 王子晶; 杜向党; 韩文瑜; 冀亚路; 雷连成; 冯新; 孙长江
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2021-08-03

Abstract

The invention discloses a genetically engineered lyase (Ply 1228) for specifically killing streptococcus suis and a preparation method thereof, and provides a novel streptococcus suis bacteriophage lyase which can be used independently or in combination with other substances, has strong lytic activity and a wider lytic spectrum, can effectively kill various serotype streptococcus suis, and provides a novel medicament for preventing and treating diseases caused by streptococcus suis infection.

Description

Gene engineering lyase for specifically killing streptococcus suis and medical application thereof

Technical Field

The invention discloses a genetically engineered lyase (Ply 1228) for specifically killing streptococcus suis and a preparation method thereof, further provides medical application of the genetically engineered lyase (Ply 1228), shows broad-spectrum efficient antibacterial activity to the streptococcus suis, and belongs to the technical field of biological engineering.

Background

Streptococcus suis is an important zoonotic pathogen. Streptococcus suis can be divided into 33 serotypes according to the antigenicity of the bacterial surface Capsular Polysaccharide (CPS). Of these,

types

2, 9, 3, 1/2 and 7 are the most prominent serotypes of streptococcus suis, and type 2 streptococcus suis is most widely distributed and most harmful worldwide. Streptococcus suis infection can cause septicemia, meningitis, endocarditis, pneumonia, arthritis, and other diseases, and the mortality rate is very high. The drug-resistant situation of streptococcus suis is more severe due to the abuse of antibiotics, and the continuous emergence of drug-resistant and multi-drug-resistant strains makes the prevention and control of antibiotics more difficult. Under the background of current 'resistance reduction', 'resistance limitation' and 'resistance prohibition', a novel antibacterial preparation which is not easy to generate drug resistance to streptococcus suis is urgently needed to be searched.

Phage lytic enzymes are a class of hydrolases encoded by the phage genome that act on bacterial cell wall peptidoglycans. The bacteriophage lytic enzyme has wide effect on gram-positive bacteria. Compared with phages and antibiotics, phage lytic enzymes have the following advantages: 1. the lyase directly acts on bacterial cell wall peptidoglycan, which is an important structure for the survival of bacteria, and the change of the peptidoglycan can have fatal influence on the bacteria, thereby greatly reducing the possibility of the bacteria to generate resistance to the lyase; 2. the lyase is protein, can be obtained by constructing, expressing and purifying an in vitro vector, can be prepared in large quantities and has lower cost; 3. the lyase is used as an antibacterial agent to enter organisms, and no biosafety problem exists. 4. The lyase is easy to design and modify manually. Gram-positive bacteria lytic enzymes are generally modular in structure, having both a hydrolysis Catalytic Domain (CD) and a cell wall binding domain (BCD). We can construct chimeric lyases with superior performance to the parent lyases by combining CD and CBD from different sources. These advantages make the lyase have great potential for application in clinical drug-resistant and multi-drug-resistant treatment of streptococcus suis infection.

Disclosure of Invention

The invention discloses a genetically engineered lyase (Ply 1228) for specifically killing streptococcus suis, which has strong bactericidal activity and a wide host spectrum on the streptococcus suis and shows strong lytic activity on 8 serotypes (2, 3, 7, 9, 10, 12, 14 and 27) of the streptococcus suis. In addition, Ply1228 shows strong acid-base and high temperature tolerance in vitro. The lyase provides a novel medicament with high potential for preventing and treating disease infection caused by streptococcus suis infection, and has high clinical application value.

The invention discloses a gene engineering lyase Ply1228 for specifically killing streptococcus suis, and an amino acid sequence of the gene engineering lyase Ply1228 is shown in a nucleotide sequence table. QSE NO:1

The invention provides an expression vector derived from a streptococcus suis gene engineering lyase Ply1228 gene and a recombinant strain thereof.

The invention provides an expression and purification method of a genetically engineered lyase Ply1228 and general biological characteristics

The invention relates to application of a gene engineering lyase Ply1228 derived from streptococcus suis in specifically cleaving streptococcus suis.

The invention relates to application of a gene engineering lyase Ply1228 derived from streptococcus suis in preparing a medicament for preventing and controlling streptococcus suis infection.

The invention relates to application of a streptococcus suis lyase Ply1228 in killing streptococcus suis in space environments, animals and human bodies.

The invention discloses a pharmaceutical composition for killing streptococcus suis, which comprises the lyase Ply1228 described in the claims as a main active ingredient.

The composition comprises any dosage form in medicine, such as liquid preparation, freeze-dried preparation or oral solid preparation.

The invention has the positive effects that:

provides a new streptococcus suis bacteriophage lyase which can be used independently or in combination with other substances, has strong lytic activity and wider lytic spectrum, can effectively kill various serotype streptococcus suis, and provides a new medicine for preventing and treating diseases caused by streptococcus suis infection.

Drawings

FIG. 1 is a diagram showing the expression and purification of the lyase Ply1228 of the present invention;

FIG. 2 is a bacterial inhibition spot pattern formed by the lyase Ply1228 of the invention;

FIG. 3 is a graph showing the pH and temperature stability assay of the lyase Ply1228 of the present invention;

FIG. 4 is a graph of in vitro bactericidal activity assay for lyase Ply1228 of the invention.

Detailed Description

The following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1

Expression and purification of lyase

An upstream primer Ply1228-F (5'-CCGCTCGAGATGATAATCAATCTTGAA-3') and a downstream primer Ply1228-R (5'-CGCGGATCCTCAAATAGGTTCTTTTGT-3') were designed based on the lyase Ply1228 nucleotide sequence (accession No. MW 582537), and the target gene was amplified using the primers Ply1228-F and Ply 1228-R. Using restriction endonucleasesXho1 andBamH1 enzyme cutting target gene and carrier pET-15 b. The enzyme-cleaved target gene and the vector were ligated together overnight at 16 ℃ under the action of T4 DNA Ligase. The ligation product was transformed into DH5 alpha competent cells by heat shock method, and the correctly identified recombinant plasmid was transformed into BL21 competent cellsA stateful cell.

500.0 mL of glycerol pET-15b-Ply1228-BL21 was cultured to logarithmic growth phase (OD 600 = 0.6-0.8), added with 1mM IPTG, and subjected to shake-induction at 16 ℃ for 18 h. The induced bacterial suspension was collected (4 ℃, 8,000 × g, 5 min) and resuspended in an appropriate amount of Tris-Cl buffer (pH = 7.5). Then, the cells were disrupted by ultrasonication and centrifuged (4 ℃ C., 10,000 Xg, 20 min) to collect the supernatant. The protein was then purified by affinity chromatography using Ni-NTA. The collected supernatant was added to a nickel column and the sample was loaded three times. Then 20 mmol and 50 mmol of imidazole are respectively added to elute the hybrid protein, and finally 500 mmol of imidazole is added to elute the target protein. Finally, protein is concentrated, 500 mmol of target protein eluent is added into an ultrafiltration tube, centrifugation is carried out (4 ℃, 3,500 Xg, 40 min), liquid in the ultrafiltration tube is taken out, quick freezing is carried out by liquid nitrogen, and then the liquid is stored in an ultra-low temperature refrigerator at minus 80 ℃. The concentration of purified lyase Ply1228 was determined using BCA protein quantification kit. And performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the pre-induction sample, the whole bacteria, the supernatant and the purified protein sample.

As shown in FIG. 1, the results of the analyses are shown in the 1, 2, 3 and 4 wells, which represent the lysate Ply 1228-induced preculture liquid, whole bacteria, supernatant and purified sample, respectively, and the purity of the purified lyase Ply1228 is high.

The formulation of the separation gel (12%) is as follows: ddH₂O: 3.3, 30% acrylamide: 4.0, 1.5 mol/L Tris.HCl (pH8.8): 2.5, 10% SDS: 0.1, 10% Ammonium Persulfate (AP): 0.1, TEMED: 0.004.

The concentrated gel formula is as follows: ddH₂O: 6.8, 30% acrylamide: 1.7, 1.0 mol/L Tris.HCl (pH6.8): 1.25, 10% SDS: 0.1, 10% Ammonium Persulfate (AP): 0.1, TEMED: 0.01.

example 2

Lyase Ply1228 plate cleavage Activity assay

Culturing the strain SC183 to a plateau stage, uniformly coating the bacterial liquid on a solid BHI culture medium flat plate by using a coating rod, hanging and dripping 10.0 mu L of purified Ply1228 (100 mu g/mL) on the flat plate, and dripping 10.0 mu L of Tris-NaCl buffer solution beside the flat plate as a control. After the plate is dried, the plate is poured into a constant temperature incubator at 37 ℃ for culturing for 20 h, and the cracking condition of the plate is observed. The results are shown in FIG. 2, where clear plaques appear at the arrows, indicating that the lyase Ply1228 has a strong lytic activity against S.suis SC 183.

Example 3

Effect of different temperatures and pH values on the Activity of the lyase Ply1228

Strain SC183 was cultured to logarithmic growth phase (OD 600 = 0.6-0.8).

Temperature stability: the bacterial liquid resuspended by the PBS buffer solution and the protein (the final concentration is 100 mug/mL) are fully and uniformly mixed at a ratio of 1:1, and the mixture is respectively placed in a water bath kettle at 4 ℃, 12 ℃, 25 ℃, 37 ℃ and 45 ℃ for incubation for 1 h, and viable count is carried out by a double dilution method. The activity is expressed by the difference between the number of live bacteria after incubation and the initial number of live bacteria, and a higher difference indicates a stronger lytic activity. Comparing the difference of the bactericidal activity of the proteins at different temperatures.

pH stability: the pH values of Tris-NaCl buffer solutions are respectively adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 by hydrochloric acid and sodium hydroxide, and the concentration of the protein Ply1228 is diluted to 100 mu g/mL by Tris-NaCl buffer solutions with different pH values. And (3) fully and uniformly mixing the bacterial liquid resuspended by the PBS buffer solution with the protein with different pH values in a ratio of 1:1, placing the mixture in a water bath kettle at 37 ℃ for incubation for 1 h, and measuring the number of viable bacteria by using a multiple dilution method. And calculating the difference between the number of the live bacteria after incubation and the initial number of the live bacteria. Comparing the difference of protein activity at different pH values.

The results are shown in fig. 3, which shows that Ply1228 shows strong acid and alkali resistance (pH = 3-9) and high temperature resistance (4 ℃ -50 ℃) in vitro.

Example 4

Determination of the in vitro bactericidal Activity of the lyase Ply1228

Strain SC183 was cultured to logarithmic growth phase (OD)₆₀₀= 0.6-0.8), washed 3 times with sterile Tris-Nacl buffer. Purified Ply1228 proteins were added to the bacterial suspension at final concentrations of 50. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL, respectively, and an equal volume of sterile Tris-NaCl buffer was added to the negative control group, and the negative control group was incubated in a 37 ℃ water bath for 1 h. Samples were taken every 10 min and colony counts were determined by the fold dilution method. This experiment was repeated three times.

Example 5

Determination of cleavage Spectrum for lyase Ply1228

Respectively culturing 49 Streptococcus suis, 10 Staphylococcus aureus, 10 Escherichia coli, 10 Salmonella, 10 Bacillus subtilis, 10 Serratia marcescens and 10 Klebsiella pneumoniae to logarithmic growth phase (OD)₆₀₀= 0.6-0.8), and then washed three times with a sterile Tris-Cl buffer, and the initial colony number was determined by the double dilution method. The lyase Ply1228 (50. mu.g/mL) was added to the bacterial solution and the mixture was incubated at 37 ℃ for 60 min and the colony count was again determined by the fold dilution method. The reduction of bacteria after incubation and initially was calculated. As a negative control, equal volumes of PBS buffer were added to the bacterial suspension for co-incubation. This experiment was repeated three times

As shown in Table 1, the lyase Ply1228 showed specific cleavage activity against Streptococcus suis, and the cleavage spectrum was broad, with cleavage activity against all 38 tested strains of Streptococcus suis. The 38 strains of S.suis contain 8 serotypes, type 2, type 3, type 7, type 9, type 10, type 12, type 14 and type 27, respectively. Lyase Ply1228 has no lytic activity against staphylococcus aureus, salmonella coli, bacillus subtilis, serratia marcescens and klebsiella pneumoniae.

TABLE 1 cleavage enzyme Ply1228 cleavage Spectrum analysis

And (4) conclusion:

the lyase Ply1228 prepared by genetic engineering has strong bactericidal activity and wide bactericidal spectrum on streptococcus suis. Therefore, the lyase can be used for effectively preventing and treating diseases caused by streptococcus suis infection. The size of the lyase is 54 kD; the lyase can form bright bacteriostatic spots on a BHI solid plate coated with the streptococcus suis SC 183; ply1228 shows strong acid and alkali resistance (pH = 3-9) and high temperature resistance (4-50 ℃) in vitro; the lyase Ply1228 is capable of cleaving streptococcus suis of 8 serotypes, including type 2, type 3, type 7, type 9, type 10, type 12, type 14 and type 27.

Sequence listing

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gcgctaatgg caggcggtgc tatttcggca ggttgggcgg ttaacacgga gtatatgcat 180

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catgggaacg gagatgcccg caaagcaagt ttgggcaatc aatatgaacc tgtcatggca 540

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atagctggta agcatggcaa cggtgaggct cgcaagcagt cgttaggtac tgactatcca 660

gcagtccaaa agcgtgtgac tgaattgctc aaaaaacagc cctcagagcc gtcaaaaggt 720

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acagaaagca aagaggaagg agacctatct ttcaatgggg caattctcaa aaaatcggtc 840

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Met Ile Ile Asn Leu Glu Thr Ser Ile Arg Trp Met Ser Asp Arg Val

1 5 10 15

Gly Lys Val Ser Tyr Ser Met Asp Tyr Arg Asn Gly Pro Asn Ser Tyr

20 25 30

Asp Cys Ser Ser Ala Val Tyr Tyr Ala Leu Met Ala Gly Gly Ala Ile

35 40 45

Ser Ala Gly Trp Ala Val Asn Thr Glu Tyr Met His Asp Trp Leu Ile

50 55 60

Arg Asn Gly Tyr Val Leu Val Ala Glu Asn Lys Pro Phe Asn Ala Gln

65 70 75 80

Arg His Asp Val Phe Ile Trp Gly Lys Arg Gly Tyr Ser Ser Gly Glu

85 90 95

Gly Gly His Thr Gly Ile Phe Val Asp Asn Val Asn Ile Ile His Cys

100 105 110

Asn Phe Lys Arg Asn Gly Ile Thr Ile Asp Asp Tyr Asn Lys Val Ser

115 120 125

Arg Gly Met Tyr Tyr Tyr Leu Tyr Arg Pro Ala Asn Gln Pro Ser Ile

130 135 140

Ser Asn Lys Ser Leu Asp Gln Leu Val Lys Glu Thr Leu Ala Gly Val

145 150 155 160

His Gly Asn Gly Asp Ala Arg Lys Ala Ser Leu Gly Asn Gln Tyr Glu

165 170 175

Pro Val Met Ala Val Ile Asn Gly Lys Ala Thr Ala Pro Lys Lys Thr

180 185 190

Val Asp Gln Leu Ala Gln Glu Val Ile Ala Gly Lys His Gly Asn Gly

195 200 205

Glu Ala Arg Lys Gln Ser Leu Gly Thr Asp Tyr Pro Ala Val Gln Lys

210 215 220

Arg Val Thr Glu Leu Leu Lys Lys Gln Pro Ser Glu Pro Ser Lys Gly

225 230 235 240

Val Glu Val Lys Gln Pro Thr Asp Thr Lys Ile Ser Gln Thr Glu Pro

245 250 255

Pro Gly Gln Ala Thr Glu Ser Lys Glu Glu Gly Asp Leu Ser Phe Asn

260 265 270

Gly Ala Ile Leu Lys Lys Ser Val Leu Asp Val Ile Leu Ala Lys Cys

275 280 285

Lys Glu His Asn Ile Leu Pro Ser Tyr Ala Ile Thr Val Leu His Phe

290 295 300

Glu Gly Leu Trp Gly Thr Ser Ala Val Gly Lys Ala Asp Asn Asn Trp

305 310 315 320

Gly Gly Met Thr Met Thr Ser Asn Asp Leu Gln Ile Thr Arg Pro Ser

325 330 335

Gly Val Ile Val Thr Arg Gly Leu Ala Arg Pro Ser Asn Glu Gly Gly

340 345 350

Tyr Tyr Met His Tyr Ala Ser Val Asp Asp Phe Leu Thr Asp Trp Phe

355 360 365

Tyr Leu Leu Arg Ala Gly Gly Ser Tyr Lys Val Ser Gly Ala Lys Thr

370 375 380

Phe Ser Glu Ala Val Lys Gly Met Phe Lys Val Gly Gly Ala Val Tyr

385 390 395 400

Asp Tyr Ala Ala Thr Gly Tyr Glu Asn Tyr Leu Val Gly Met Ser Ser

405 410 415

Arg Leu Thr Ala Ile Glu Ser Glu Asn Gly Ser Leu Ala Lys Tyr Asp

420 425 430

Gln Gln Thr Val Thr Asp Val Ala Lys Ile Asp Lys Ile Glu Val Ala

435 440 445

Ile Glu Gly Ile Glu Val Thr Ile Asn Gly Thr Arg Tyr Arg Leu Thr

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Lys Glu Pro Ile

465

Claims

1. A genetically engineered lyase Ply1228 for specifically killing Streptococcus suis, the amino acid sequence and nucleotide sequence of the genetically engineered lyase are as follows: shown as SEQ No. 1.

2. An expression vector comprising the genetically engineered lyase Ply1228 gene of claim 1.

3. Construction of a recombinant strain comprising the expression vector for the lyase Ply1228 according to claim 1.

4. The method for expressing and purifying the engineered lyase Ply1228 of claim 1, wherein the lyase primers are amplified by the following sequences:

upstream primer Ply 1228-F: 5'-CCGCTCGAGATGATAATCAATCTTGAA-3', respectively;

downstream primer Ply 1228-R: 5'-CGCGGATCCTCAAATAGGTTCTTTTGT-3' are provided.

5. The preparation method of the genetically engineered lyase Ply1228 of claim 1, comprising the steps of:

1) primer design of the lyase nucleotide sequence of claim 2;

2) cloning the lyase nucleotide sequence of claim 2 into an expression vector to obtain a recombinant plasmid;

3) transferring the recombinant plasmid obtained in the step 1 into a strain to obtain a recombinant strain;

4) inducing and expressing the lyase by using a recombinant strain;

5) the lyase obtained in step 3 is further purified.

6. Use of the genetically engineered lyase Ply1228 according to claim 1 for the preparation of a medicament for the prevention and treatment of infectious diseases in animals and humans caused by streptococcus suis.

7. A composition for preventing and treating infectious diseases caused by streptococcus suis, comprising the genetically engineered lyase Ply1228 of claim 1 as an effective ingredient.

8. The composition of claim 7 comprises any dosage form of liquid, lyophilized or oral solid.