CN105085675A - Mouse SPATA16 gene polyclonal antibody and preparation method thereof - Google Patents

Abstract

The invention discloses a mouse SPATA16 gene polyclonal antibody and a preparation method thereof. The method comprises following steps: a) amplifying SPATA16 gene sequences indicated by SEQ ID NO:1 and pET32a expression plasmids indicated by SEQ ID NO:2 by a PCR method; b) connecting the SPATA16 gene with a pET32a expression vector to constitute a recombinant plasmid; c) transferring the recombinant plasmid into BL21 competent bacteria, after culturing, inducing, cracking and purifying, obtaining specific target proteins; and d) immunizing a rabbit, collecting blood, purifying antibody serum, and obtaining polyclonal antibodies. The polyclonal antibody of SPATA16 gene is prepared to recognize a target protein, so that research of expression and positioning of target proteins is facilitated.

Description

Mouse SPATA16 gene polyclonal antibody and preparation method thereof

Technical field

The application relates to a kind of mouse SPATA16 gene polyclonal antibody and preparation method thereof.

Background technology

SPATA16 gene is a new gene, and research shows, it has certain function in mouse sperm generating process, therefore, is necessary further to study it.

Summary of the invention

The invention provides a kind of new mouse SPATA16 gene polyclonal antibody and preparation method thereof.

The invention provides a kind of preparation method of mouse SPATA16 gene polyclonal antibody, comprise the steps:

A) with SPATA16 gene as shown in SEQIDNO:1 of PCR method extension increasing sequence and the pET32a expression plasmid of sequence as shown in SEQIDNO:2;

B) SPATA16 gene is connected to construction recombination plasmid in pET32a expression vector;

C) described recombinant plasmid is proceeded to BL21 competence bacterium, through cultivation, induction, cracking and purifying, obtain specificity target protein;

D), after immune rabbit, blood sampling, antibody purification serum, obtains polyclonal antibody.

Described step a) in, during pcr amplification, SPATA16 upstream region of gene primer is as shown in SEQIDNO:3, and SPATA16 downstream of gene primer is as shown in SEQIDNO:4; PET32a plasmid upstream primer is as shown in SEQIDNO:5, and pET32a plasmid downstream primer is as shown in SEQIDNO:6.

Described step c) in, during cultivation, the bacterium liquid containing described recombinant plasmid is joined in autoclaved antibiotic-free LB substratum, and adds ammonia benzyl microbiotic.

Described step c) in, induced by IPTG.

Described step c) in, utilize lysate to carry out cracking, described lysate comprises TrisHCl, NaCl and EDTA.

Described step c) in, described purifying comprises inclusion body protein process and gradient dialysis.

A kind of mouse SPATA16 gene polyclonal antibody, the protein sequence of described antibody recognition is as shown in SEQIDNO:7.

The invention has the beneficial effects as follows: by preparing the polyclonal antibody of gene SPATA16, this antibody can specific recognition target protein, thus is convenient to the expression and location etc. of research purpose albumen.

Accompanying drawing explanation

Fig. 1 is SPATA16 clone identification figure, and wherein, it is 450bp that SPATA16 intercepts clip size; Clip size and spata16 in the same size;

Fig. 2 is the electrophorogram after NovagenIDA-Ni resin purification His-Spata16-150aa protein purification;

Fig. 3 is the expression characteristic figure of SPATA16 in different Testicular Cells system, and wherein, 1 represents mouse testis total protein; 2 represent TM4 clone (Properties of Sertoli Cells Isolated from Mice Testis); 3 represent CRL-2053 clone (Mouse Spermatogonial Cells); 4 represent CRL-2196 clone (mouse spermatocyte);

Fig. 4 is the Subcellular Localization figure of SPATA16 at 293T; In 293T cell, SPATA16 is mainly positioned in nucleus;

Fig. 5 is the Subcellular Localization figure of SPATA16 in mouse testis tissue; In mouse tissue, in karyon, expression signal intensity is much larger than endochylema, shows that SPATA16 is positioned mouse testis histocyte core;

Fig. 6 is SPATA16-150aa antibody test mSPATA16 Subcellular Localization figure, and wherein, mSPATA16 is mainly expressed in the nucleus of HEK293 cell.

Embodiment

By reference to the accompanying drawings the present invention is described in further detail below by embodiment.

In one embodiment, the preparation method of mouse SPATA16 gene polyclonal antibody comprises the steps:

A) with SPATA16 gene as shown in SEQIDNO:1 of PCR method extension increasing sequence and the pET32a expression plasmid of sequence as shown in SEQIDNO:2; Wherein, SPATA16 gene order is existing sequence, and its log-on message is: NM_027583, and the protein sequence of this gene is as shown in SEQIDNO:8, and its accession number is: NP_081859.

B) SPATA16 gene is connected to construction recombination plasmid in pET32a expression vector; During connection, T4 ligase enzyme can be adopted.

C) described recombinant plasmid is proceeded to BL21 competence bacterium, through cultivation, induction, cracking and purifying, obtain specificity target protein.

D), after immune rabbit, blood sampling, antibody purification serum, obtains polyclonal antibody.

As shown in Figures 1 to 6, in another embodiment, the preparation method of mouse SPATA16 gene polyclonal antibody comprises the steps:

1, by not relying on cloning process (LIC) recombinant plasmid of ligase enzyme

A) design the primer of plasmid pET32a and gene SPATA16mRNA, primer is as shown in table 1:

The synthetic primer of table 1 plasmid pET32a and gene SPATA16

B) by the method amplification object fragment SPATA16 and expression plasmid pET32a (adopting the PrimeSTARGXLDNAPolymerase of TAKARA company) of PCR, GXL High fidelity PCR reaction system is as shown in table 2:

Table 2:PCR reaction system

Reagent	Usage quantity
		5 × PrimeSTAR GXL damping fluid	10μl
dNTP Mixture(2.5mM each)	4μl
		Primer F (SPATA16 and pET32a upstream primer)	10～5pmol
Primer R (SPATA16 and pET32a downstream primer)	10～15pmol
		Template (template DNA)	100ng
PrimeSTAR GXL DNA Polymerae	1μl
		Sterile purified water	Up to50μl

PCR reaction conditions is: 1.98 DEG C of 5min; 2,98 DEG C of 10sec; 3,60 DEG C of 15sec; 4,68 DEG C of 17min; 5,2 ~ 4 circulation 30 times; 6,60 DEG C of 10min; 7,12 DEG C of O/N (overnight spends the night)

In PCR reaction system, dNTPMixture is the mixing solutions of dATP, dCTP, dGTP and dTTP, and the concentration of often kind is 2.5mM

C) by gel purification kit purifying gained SPATA16 fragment and pET32a expression vector.

D) T4DNA ligase enzyme (TAKARAT4DNAPolymerase2040A) processes pET32a expression vector and SPATA16 fragment.

D1) every 20ul reaction solution adds the pET32a expression vector of 0.037pmol;

D2) every 20ul reaction solution adds the glue recovery purified product of 0.12-0.48pmol;

D3) by T4 ligase enzyme process pET32a expression vector and SPATA16 fragment, its reaction system is as shown in table 3;

Reaction system during table 3T4 ligase enzyme process

a:Vector Mix	1X
		T4Buffer (damping fluid)	2μl
DTT(100mM)	1μl
		T4Polymerase (T4 ligase enzyme)	0.4μl
dGTP	2μl
		ddH2O	fill to make20ul
b:Insert Mix	1X
		T4Buffer	2μl
DTT(100mM)	1μl
		T4Polymerase	0.4μl
dCTP	2μl
		ddH2O	fill to make20μl

D4) under 22 DEG C of conditions, 30min is hatched;

D5) 20min is hatched with deactivation T4 ligase enzyme for 75 DEG C.

E) expression vector is connected with object fragment

E1) by sample (mixed solution of expression vector and object fragment) centrifugal treating;

E2) according to the concentration of the gained of expression vector and object fragment, both are mixed in a pipe with the copy number of 1:1;

E3) 5min is hatched for 22 DEG C;

E4) add 2.5 μ lEDTA (25mM) and mix;

E5) 5min is hatched for 22 DEG C;

E6) constructed recombinant plasmid is proceeded to BL21 competence bacterium.

2, expressed fusion protein pET32a-SPATA16

A) select the mono-clonal after conversion in 5MLLB substratum, 37 DEG C, 280rpm incubator shakes bacterium 16h.

B) the antibiotic-free LB substratum of 100ml is loaded with 500ML Erlenmeyer flask, autoclaving.

C) the 1ml bacterium liquid containing recombinant plasmid is joined in autoclaved antibiotic-free LB substratum, add the ammonia benzyl microbiotic of 100ul concentration 100ug/ml.

D) 37 DEG C, 280rpm shakes bacterium after two hours 45 minutes, detects its OD ₅₉₅≈ 0.6.

E) cooled on ice 15 minutes.

F) IPTG (original content 24mg/ml, final concentration 1/100) is added.

G) 37 DEG C, 280rpm shakes bacterium three hours.

H) 12000rpm, 4 DEG C, centrifugal 30min.Collect heavy bacterium, claim net weight.

I) every 1g adds 2 ~ 5mlbufferW (100MmTrisHClph8.0,100mMNaCl, 1mMEDTA), piping and druming mixing, in power 17% ~ 20% ultrasonication, is advisable to suspension clarification.

J) 12000rpm, 4 DEG C, centrifugal 10min.Collect supernatant liquor and precipitation.

3, inclusion body protein process:

A) urea (the being melted into 1XPBS) precooling on ice of 8M is configured.

B) the heavy bacterium after ultrasonication is melted in 8M urea, piping and druming mixing, 4 DEG C of vertical rotary 8h ~ O/N.

C) 4 DEG C, the centrifugal 15min of 12000rcf, collect supernatant, supernatant is soluble protein.

D) add 10mM imidazoles with the 8M urea of two volumes and dissolve soluble protein, add the beadz of pre-prepd band specific label.

E) 4 DEG C are rocked 1 ~ 2h.

F) the centrifugal 10min of 10000rcf.

G) beadz is collected with prepacked column.

H) with 8M urea preparation 300mM imidazoles, the target protein being adsorbed on beadz is eluted.

4, gradient dialysis:

A) 7M, 6M, 5M, 4M2M, 0M urea is prepared respectively, precooling with 1XPBS.

B) assemble semi-permeable membranes dialysis tubing, target protein is sealed in dialysis tubing, dialyse from the urea of the urea of high density concentration on earth respectively, each concentration dialysis 4 ~ 6h.

C) reclaim protein solution, carry out ultrafiltration and concentration with super filter tube.

D) finally by protein concentrate-80 DEG C maintenance, use in order to subsequent experimental.

Above-mentioned gradient dialysis and inclusion body protein process are all for purifying protein.

5, obtain specificity target protein, subcutaneous injection enters in rabbit body.

A) 1.5mmSDS-PAGE albumin glue some pieces is prepared.

B) protein solution is carried out SDS-PAGE and run glue analysis.

C) prepare 2MKCl500ml, hatch on ice.

D) albumin glue is soaked into completely preprepared iced KCL solution, after shaking table rocks 5min, can sees near object stripe size, have special white ribbon occur.Cut after being defined as object band, be collected in the centrifuge tube of 4ml.

E) dissolve with 400 ~ 500ul1XPBS, the albumin glue grinding pulping that agitator or homogenizer will cut down.

F) slurries are collected.

G) with Y-tube syringe, slurries and adjuvant are mixed with the ratio of 1:1.To see that oyster white mixing liquid is advisable.

H), after fully mixing, collect mixed solution, be injected in rabbit body.Each 2ml, once in a week, continues 1.5 months.Rabbit is new zealand white rabbit.

I) after two months, rabbit ear vein blood is extracted.Ambient temperature overnight.Extract the polyclonal antibody that serum is this target protein.

J) Identification of the antibodies is carried out with westernblot.

Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made.

Claims (7)

1. a preparation method for mouse SPATA16 gene polyclonal antibody, is characterized in that, comprise the steps:

A) with SPATA16 gene as shown in SEQIDNO:1 of PCR method extension increasing sequence and the pET32a expression plasmid of sequence as shown in SEQIDNO:2;

B) SPATA16 gene is connected to construction recombination plasmid in pET32a expression vector;

C) by described recombinant plasmid transformed in BL21 competence bacterium, through cultivating, induction, cracking and purifying, obtain specificity target protein;

D), after immune rabbit, blood sampling, antibody purification serum, obtains polyclonal antibody.

2. the preparation method of mouse SPATA16 gene polyclonal antibody as claimed in claim 1, it is characterized in that, described step a) in, during pcr amplification, SPATA16 upstream region of gene primer is as shown in SEQIDNO:3, and SPATA16 downstream of gene primer is as shown in SEQIDNO:4; PET32a plasmid upstream primer is as shown in SEQIDNO:5, and pET32a plasmid downstream primer is as shown in SEQIDNO:6.

3. the preparation method of mouse SPATA16 gene polyclonal antibody as claimed in claim 1, it is characterized in that, described step c) in, during cultivation, bacterium liquid containing described recombinant plasmid is joined in autoclaved antibiotic-free LB substratum, and adds ammonia benzyl microbiotic.

4. the preparation method of mouse SPATA16 gene polyclonal antibody as claimed in claim 1, is characterized in that, described step c) in, induced by IPTG.

5. the preparation method of mouse SPATA16 gene polyclonal antibody as claimed in claim 1, is characterized in that, described step c) in, utilize lysate to carry out cracking, described lysate comprises TrisHCl, NaCl and EDTA.

6. the preparation method of mouse SPATA16 gene polyclonal antibody as claimed in claim 1, is characterized in that, described step c) in, described purifying comprises inclusion body protein process and gradient dialysis.

7. a mouse SPATA16 gene polyclonal antibody, is characterized in that, the protein sequence of described antibody recognition is as shown in SEQIDNO:7.