CN101830983B

CN101830983B - Preparation and application of specific sequence-based aquaporin 4 high titer antibody

Info

Publication number: CN101830983B
Application number: CN2010101648511A
Authority: CN
Inventors: 李晓萌; 张淑芝; 汪小莞; 关新刚; 杨南扬; 麻彤辉
Original assignee: Northeast Normal University
Current assignee: Northeast Normal University
Priority date: 2010-05-07
Filing date: 2010-05-07
Publication date: 2012-11-07
Anticipated expiration: 2030-05-07
Also published as: CN101830983A

Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to the preparation and application of a specific sequence-based aquaporin 4 high titer antibody. By adopting the gene engineering technology, the 3' end proximal hydrophilic area of the AQP4 gene of a mouse is cloned in a prokaryotic expression vector; after enzyme digestion and sequence analysis, Escherichia coli is converted by recombinant plasmids and generates GST-AQP4 fusion proteins by induction of isopropyl-beta-D-thiogalactoside; the purified fusion proteins are used for immunizing New Zealand rabbit to prepare antiserum; the titer and the specificity of the antiserum are detected by ELISA and Western bolt; and by constructing the prokaryotic expression vector of the fusion proteins, the specific fusion proteins can be expressed in high efficiency to form high titer antiserum of resisting the GST-AQP4 fusion proteins. The AQP4 poly-cloning antibody is high in the titer as well as specificity and is applied to the detection of the AQP4. The invention provides a necessary experimental device for deep researches on the AQP4 function at the protein level.

Description

The preparation and the application of aquaporin 4 high-titer antibodies on distinguished sequence basis

Technical field

The invention belongs to the DNA recombinant technology field in the biotechnology, be specifically related to the polyclonal antibody and the application of the prokaryotic expression GST-AQP4 fusion rotein on distinguished sequence basis.

Background technology

(aquaporin AQP) is one type and extensively is present on protokaryon and the eukaryotic cell membrane aquaporin, and at least 12 members have been found at mammals at present in the special duct of selectivity efficient transhipment water molecules.Research shows, AQP family plays a significant role in the fluid transport physiology of multiple tissue and pathology.Aquaporin AQP4 locates high expression level at the collecting tubule of stellate cell, eyes, ear, Skelettmuskel, gastral cavity parietal cell and the kidney of brain etc., and osmotic pressure drive stride in the severe edema due to hypofunction of the spleen play a significant role in transporting.

Since two scientists of the U.S. in 2003 obtained Nobel chemistry Prize then because of some the initiative work in the membrane channel research field, the research of aquaporin structure and function had become focus.For a kind of new protein in fact, antibody is one of the strongest instrument of its function of research.The a series of technology that on the antigen-antibody interaction basis, grow up like immunohistochemistry, immunoblotting and immunoprecipitation etc., all have a wide range of applications in proteinic detection and functional study.

Aquaporin 4 is as a kind of protein molecular of striding film, and hydrophobic fragment wherein can't be carried out prokaryotic expression, and outer little hydrophilic region no antigen or antigenicity of film is less.The AQP4 antibody that this life, face was sold is to form as antigen prepd with the synthetic short peptide chain, and the space structure of its antigen protein has very big-difference than the proteic space structure of AQP4.

Summary of the invention

The objective of the invention is to set up the model of the detection of a kind of AQP4 of can be used in, for necessary experimental tool being provided from protein level further investigation AQP4 function and relative disease.

Utilize genetic engineering technique, the nearly 3 ' end of mouse AQP4 gene hydrophilic area is cloned among the prokaryotic expression carrier pGEX-4T-1.Through enzyme cut with sequential analysis after, use the recombinant plasmid transformed e. coli bl21, and induce through isopropyl-(IPTG) and to produce the GST-AQP4 fusion rotein.Fusion protein immunization new zealand rabbit with purifying prepares antiserum(antisera).Sero-fast tiring and specificity employing ELISA and Western blot detection.

GST-AQP4 fusion rotein of the present invention is with utilizing special sequence (the nearly 3 ' end of mouse AQP4 gene hydrophilic area) to make up GST-AQP4 fusion protein prokaryotic expression carrier; Transform the specific GST-AQP4 fusion rotein that efficiently expresses that BL21 obtains, the nearly 3 ' end of this section hydrophilic area gene nucleotide series is:

781 tgtcctgatg?tggagctcaa?acgtcgcctt

841aaggaagcct?tcagcaaagc?cgcgcagcag?acaaaaggga?gctacatgga?ggtggaggac

901aaccggagcc?aagtggagac?ggaagacttg?atcctg

Expressed proteic aminoacid sequence is:

tkgsymev?ednrsqvete?dlilkpgvvh?vididrgeek?kgkd

This GST-AQP4 fusion rotein and polyclonal antibody preparation thereof may further comprise the steps:

The first step, the structure of cloning vector

Using P CR technology is template with p8p64 from the mouse AQP4 full length gene, and the purpose fragment that amplification obtains is connected with the PMD18-T carrier, and after steps such as conversion, extraction obtained recombinant plasmid, enzyme was cut evaluation and checked order.

Second step, the structure of prokaryotic expression carrier pGEX-4T-1

Behind cloned plasmids and plasmid pGEX-4T-1 double digestion, utilizing the recovery test kit to obtain the AQP4 gene should district's fragment be connected with carrier.Obtain the expression vector of reorganization through steps such as conversion, extractions.Enzyme is cut the evaluation recombinant chou, and order-checking is further confirmed.

The 3rd step, the abduction delivering of GST-AQP4 fusion rotein and purifying

Recombinant plasmid PGEX-4T-1/AQP1 transformed into escherichia coli BL21 utilizes IPTG to induce, the GST-AQP4 Expression of Fusion Protein.Identify with SDS-PAGE, and the optimization expression condition, increase in a large number and induce.Use the ultrasonic degradation bacterium, the institute albumen that obtains with Glutathione-Sepharose 4B column purification, SDS-PAGE purification Identification product.

In the 4th step, the sero-fast preparation of the anti-AQP4 of rabbit promptly obtains the polyclonal antibody of this invention

With the male new zealand white rabbit of AQP4 fusion protein immunization of purifying, initial immunity is with 500 μ g fusion roteins, with the subcutaneous multi-point injection of the abundant mixing emulsification of isopyknic complete Freund's adjuvant back part.Get the ear vein blood system before the immunity from serum, as the contrast of the serum before the immunity.Carry out booster immunization behind the 2wk the 1st time, the GST-AQP4 fusion rotein of 500 μ g purifying and incomplete Freund's adjuvant equal-volume mixing, front and back four sole intramuscular injection.Every afterwards separated 2wk booster immunization 1 time.1wk gets ear blood after the last immunity, measures tiring of antibody with the ELISA method, and when antibody titer reached 1: 100000, serum was collected in the carotid artery bloodletting, and it is tired and specificity to use the detection of panimmunity method.

We use this and carry out the immunohistochemical methods evaluation, promptly get the wild-type mice brain, knock out the mouse brain with AQP4 and do contrast, do immunohistochemical methods.Tissue shredded add an amount of RIPA lysate [0.1%SDS, 150Mm NaCl, 10Mm Tris, (pH7.4), 1%Triton X-100,1% sodium deoxycholate, 1mM PMSF], after fully grinding, centrifugal collection supernatant.The total protein of cell of getting equivalent carries out the 12%SDS-PAGE electrophoretic separation, and electrotransfer is to pvdf membrane.5% skim-milk sealing 1h; With homemade I level antibody room temperature reaction 1h, PBS washes 3 times; With goat-anti rabbit II level antibody (Jackson Immunoresearch Laboratories) the room temperature reaction 1h of HRP mark, PBS washes 3 times, with ECL Photochemical agents box (Amersham Phamacia) detection signal, X-ray sheet exposure imaging again.The result shows that the anti-GST-AQP4 serum of this rabbit can be specific and the AQP4 protein binding, successfully AQP4 has been carried out detecting [Fig. 7].

3 ' end hydrophilic area gene order that utilization is chosen successfully makes up GST-AQP4 fusion protein prokaryotic expression carrier, can efficiently express specific GST-AQP4 fusion rotein behind the conversion BL21.Tire antiserum(antisera) and specificity of the height that obtains anti-GST-AQP4 fusion rotein with this fusion protein immunization rabbit is good; When having overcome existing antibody with artificial synthesis peptide's preparation; Its antigen space structure and the big shortcoming of expressing protein difference, tire and the specificity of resulting antibody all has significantly lifting.Can be applicable to detection, advance further investigation from protein level AQP4 function to AQP4.

Description of drawings:

Nearly 3 ' the end of the AQP4 gene hydrophilic area DNA agarose gel electrophoresis figure that Fig. 1 goes out for pcr amplification;

Fig. 2 is double digestion plasmid pGEX-4T-1 agarose gel electrophoresis figure;

Fig. 3 is a pGEX-4T-1/AQP4 recombinant chou sequencing result;

Fig. 4 is GST and the GST-AQP4 fusion rotein SDS-PAGE figure behind the purifying, wherein 1,2.GST (26KD); 3,4.GST-AQP4 (30KD);

Fig. 5 measures tiring of antibody for indirect elisa method;

Fig. 6 is that the specific Western blot of antiserum(antisera) analyzes, wherein 1,2, and 4. wild-type CD1 mouse brain tissue; 3.buffer contrast;

Fig. 7 is anti-GST-AQP4 seroimmunity group qualification result, and wherein the left side is the cerebral tissue of wild mouse, and the right side is the cerebral tissue that knocks out mouse.

Embodiment:

Embodiment 1: anti-GST-AQP4 serum the preparation

1.PCR-PMD18-T/AQP4 construction of recombinant plasmid

Mouse AQP4 full length gene obtains from Genebank, and gene order number is NM009700.With p8p64 is template, and upstream primer is 5 '-GAATTCGACAACCGGAGCCAAGTG (containing the EcoR1 restriction enzyme site); Downstream primer is 5 '-CTCGAGTACGGAAGACAATACCTC (containing the XhoI restriction enzyme site).Using P CR has successfully amplified the nearly 3 ' end of AQP4 gene hydrophilic area dna sequence dna, length 102bp, 34 amino acid [Fig. 1] of encoding.The fragment that obtains of amplification is connected with the PMD18-T carrier, will connect product and change in the competence bacillus coli DH 5 alpha, is containing selected clone on the Amp+ agar plate, carry recombinant plasmid for a short time with alkaline lysis after, with EcoR I and the evaluation of Xhol I double digestion.

2. the structure of prokaryotic expression carrier pGEX-4T-1

To contain the segmental pMD18-T plasmid of AQP4 gene nearly 3 ' end hydrophilic area behind EcoR I and Xho1 double digestion, and utilize the recovery test kit to obtain the AQP4 gene and be somebody's turn to do district's fragment, handle plasmid pGEX-4T-1 [Fig. 2] with identical enzyme simultaneously.Then the nearly 3 ' end of the AQP4 gene that reclaims hydrophilic area fragment and the carrier pGEX-4T-1 that cuts through enzyme are connected in 16 ℃ under the effect of T4 dna ligase and spend the night.Enzyme is cut the evaluation recombinant chou, and order-checking further definite [Fig. 3], and the result shows that this AQP4 fragment is correct.

3.GST-AQP4 the abduction delivering of fusion rotein and purifying

Recombinant plasmid PGEX-4T-1/AQP1 transformed into escherichia coli BL21, picking list bacterium colony insert in the LB/Ampr substratum, 37 ℃ of jolting overnight cultures.Next day, culture is transferred in the LB substratum that contains Amp+ in 1: 50 ratio, continue to be cultured to logarithmic growth mid-term at 37 ℃ of shaking tables.At the A600 of nutrient solution is 0.5～0.6 o'clock, add IPTG to final concentration be 0.08mmol/L, do not add the negative contrast of IPTG person, put 25 ℃ and continue to cultivate 4～5h.Centrifugal collection thalline is identified the GST-AQP4 Expression of Fusion Protein with SDS-PAGE, and the optimization expression condition, increases in a large number and induces.In 4 ℃ of centrifugal 5min, collect thalline with 5000r/min, ice the deposition of the NETN suspension 1L bacterium liquid of precooling with 60mL.Use the ultrasonic degradation bacterium, again with 9600rpm, in 4 ℃ of centrifugal 15min; Get supernatant, cross Glutathione-Sepharose 4B post, (contain 20mM gsh, 50mMTriscl with equal-volume elution buffer 1 earlier; PH=8.0) wash-out is collected elutriant, washes twice with equal-volume elution buffer 2 (containing the 100mM gsh) again; Collect elutriant, SDS-PAGE purification Identification product [Fig. 4].

1, sero-fast preparation of the anti-AQP4 of rabbit and analysis

I. sero-fast preparation

With the male new zealand white rabbit of AQP4 fusion protein immunization of purifying, initial immunity is with 500 μ g fusion roteins, with the subcutaneous multi-point injection of the abundant mixing emulsification of isopyknic complete Freund's adjuvant back part.Get the ear vein blood system before the immunity from serum, as the contrast of the serum before the immunity.Carry out booster immunization behind the 2wk the 1st time, the GST-AQP4 fusion rotein of 500 μ g purifying and incomplete Freund's adjuvant equal-volume mixing, front and back four sole intramuscular injection.Every afterwards separated 2wk booster immunization 1 time.1wk gets ear blood after the last immunity, measures tiring of antibody with the ELISA method, and when antibody titer reached 1: 100000, serum was collected in the carotid artery bloodletting.

Ii. the mensuration of antiserum titre

As contrast, get behind the last booster immunization the 7th day serum with the new zealand white rabbit serum before the GST-AQP4 fusion protein immunization.10 times of serum dilutions earlier behind the doubling dilution, are measured tiring of antibody with indirect ELISA again.The result shows that the rabbit anteserum before the immunity is not measured the antibody of anti-fusion rotein GST-AQP4, after the last immunity in the antiserum(antisera) titre of anti-GST-AQP4 antibody up to 1: 50000 above [Fig. 5].

The specific Western blot of iii antiserum(antisera) analyzes

Carry out Western blot with soluble g ST-AQP4 fusion rotein as the anti-GST-AQP4 serum of the rabbit of immunogen preparing, with the fusion rotein GST2LZP3 sample of purifying, after SDS-PAGE separates again electrotransfer to nitrocellulose filter.With 5% skim-milk sealing 1h, drip the anti-mouse LZP3 of rabbit antiserum(antisera) (room temperature 2h, PBS wash 3 times) and goat anti-rabbit igg 2HRP (room temperature reaction 1h, PBS washing 3 times) successively, add substrate DAB colour developing at last, and take pictures.In Mr * 103 is that 1 special protein band appears in 30 places, explains that anti-GST-AQP4 serum has specificity [Fig. 6] preferably.

Embodiment 2: the application of anti-GST-AQP4 serum

Use this and carry out the immunohistochemical methods evaluation, promptly get the wild-type mice brain, knock out the mouse brain with AQP4 and do contrast, do immunohistochemical methods.Tissue shredded add an amount of RIPA lysate [0.1% SDS, 150Mm NaCl, 10Mm Tris, (pH7.4), 1%Triton X-100,1% sodium deoxycholate, 1mM PMSF], after fully grinding, centrifugal collection supernatant.The total protein of cell of getting equivalent carries out the 12%SDS-PAGE electrophoretic separation, and electrotransfer is to pvdf membrane.5% skim-milk sealing 1h; With homemade I level antibody room temperature reaction 1h, PBS washes 3 times; Again with goat-anti rabbit II level antibody (JacksonImmunoresearch Laboratories) the room temperature reaction 1h of HRP mark; PBS washes 3 times; With ECL Photochemical agents box (AmershamPhamacia) detection signal; X-ray sheet exposure imaging, the result shows that the anti-GST-AQP4 serum of this rabbit can be specific and the AQP4 protein binding, successfully AQP4 has been carried out detecting [Fig. 7].

Fig. 1

Fig. 2

Claims

1. the polyclonal antibody of the prokaryotic expression GST-AQP4 fusion rotein on distinguished sequence basis; It is characterized in that utilizing the GST-AQP4 fusion rotein of distinguished sequence prokaryotic expression to produce antiserum(antisera) and obtain as antigen-immunized animal; The titre of anti-GST-AQP4 antibody was up to 1: 50000 in this antiserum(antisera); And identify that through Western blot and immunohistochemical methods anti-GST-AQP4 serum has specificity preferably; Wherein, described distinguished sequence is meant one section sequence of the nearly 3 ' end of AQP4 gene hydrophilic area, and its aminoacid sequence is:

tkgsymev?ednrsqvete?dlilkpgvvh?vididrgeek?kgkd，

Nucleotides sequence is classified as:

781 tgtcctgatg?tggagctcaa?acgtcgcctt

841aaggaagcct?tcagcaaagc?cgcgcagcag?acaaaaggga?gctacatgga?ggtggaggac

901aaccggagcc?aagtggagac?ggaagacttg?atcctg。