CN102286068A

CN102286068A - Synergistic protein for rabies virus vaccine, gene for encoding protein and application of protein

Info

Publication number: CN102286068A
Application number: CN2011102034403A
Authority: CN
Inventors: 朱乃硕; 胡晓波
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2011-07-20
Filing date: 2011-07-20
Publication date: 2011-12-21

Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to a synergistic protein for rabies virus vaccine, a gene for encoding the protein and application of the protein. cDNA (complementary deoxyribose nucleic acid) of the protein is cloned to procaryotic cell and eukaryotic (yeast, animal and plant) cell by a genetic engineering method so as to express the protein encoded by the cDNA. The protein has high compatibility to the rabies virus vaccine. When the protein is cooperatively used with the rabies virus (antigen) vaccine, the time of generating a rabies virus (antigen) antibody can be obviously accelerated, the immune effect of the vaccine is improved, and the titer of a protective antibody in a body of an animal is improved. The protein disclosed by the invention can be used as an adjuvant of the rabies virus vaccine for developing the high-efficiency rapid synergism-type rabies virus vaccine, so that the effect on emergency prevention and treatment protection after the animal is infected with viruses is achieved.

Description

A kind of hydrophobia synergistic protein and coding this proteic gene and application

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of hydrophobia synergistic protein and coding this proteic gene and application.

Background technology

Rabies are a kind of more serious acute infectious diseases of current threat human health, and in a single day human patients is bitten by ill domestic animal and fall ill, and mortality ratio is up to more than 95%.Current China rabies situation is also quite severe, and sickness rate occupies the second place of the world, is only second to India.By the antirabic infection of injection hydrophobia is the important channel of current control rabies morbidity.But the immune effect of hydrophobia exists in the crowd than big-difference at present; and the titre of rabies virus antibodies rises to have and protects the needed time of rendeing a service of titre longer in the body of injection back, is not enough to produce infection and the morbidity of enough antibody with the opposing rabies virus in the phase in virus lays dormant.Therefore seeking the molecule that can effectively strengthen the hydrophobia immunogenicity is an important channel that addresses this problem as adjuvant.

Adopt Principles of Gene Engineering, using protokaryon (as intestinal bacteria) expression system or certain albumen of eucaryon (as yeast, mammalian cell etc.) expression system clonal expression is to obtain a large amount of these proteic effective meanss the most.With itself and hydrophobia immune animal together, measure the antibody titer of rabies virus specific antigens in the animal body again with the ELISA method, can observe this albumen easily and whether can strengthen the immune effect of hydrophobia, thereby infer whether this albumen has the potential using value in the hydrophobia field.

Summary of the invention

The purpose of this invention is to provide synergistic protein and this proteic gene of coding of a kind of hydrophobia, and provide expression these proteic methods and applications.

The synergistic protein of hydrophobia provided by the invention (being designated as SBP), its nucleotides sequence is classified as shown in the SEQ.ID.NO.1, has following functional character: 1. can be affine with hydrophobia, record affinity costant and can reach 1.16 * 10 ⁷L/mol; 2. with hydrophobia injection animal or human body, the time of the antibody titers that reaches protection effectiveness be can shorten, immunne response efficient and intensity improved.

The present invention also provides the gene of the synergistic protein of the described hydrophobia of coding, and its aminoacid sequence is shown in the SEQ.ID.N0.2.

By genetic engineering modified, the present invention has also obtained a series of homologys and has reached the sequence that hydrophobia is had synergistic function that has more than 80%.Specifically comprise following sequence:

1, obtains some gene orders that have with the same immunopotentiation function of SBP by amplification in vitro splicing or DNA synthetic, gene order number is SEQ.ID.NO.3--SEQ.ID.NO.5, through the protein sequence of modifying: SEQ.ID.NO.6, SEQ.ID.NO.7.

2, several protein sequences have been obtained by means such as rite-directed mutagenesis, gene recombination: SEQ.ID.NO.8, SEQ.ID.NO.9 with stronger affinity immune synergistic function.

3, reach 80% or more with said gene or protein sequence (SEQ.ID.NO.1--SEQ.ID.NO.9) homology and have to reach with the hydrophobia high affinity hydrophobia is had the sequence of remarkable synergistic function by what the whole bag of tricks obtained, above-mentioned sequence is referred to as SBPs.

Above-mentioned dna sequence dna/fragment directly is cloned into pBV220 or other prokaryotic expression carrier, obtains recombinant protein through abduction delivering; Also can (a, b c) wait and express in mammalian cell and yeast to carrier for expression of eukaryon such as pEGFP-N1, pICZ α with above-mentioned expression fragment cloning.The affinity column of utilization Ni-NTA agarose, the crosslinked SPA of having, ConA, mAb etc. carries out purifying, finally obtains the target protein of purifying.

The albumen that nucleotides sequence of the present invention is classified SEQ.ID.NO.1 as can be used as the hydrophobia adjuvant.This albumen and hydrophobia have high-affinity; can significantly quicken the time that rabies virus (antigen) antibody produces with the coupling of rabies virus (antigen) vaccine; improve the immune effect of vaccine; improve protection antibody titre in the animal body, emergence control is protected after being used in animal and being subjected to virus infection.

Aminoacid sequence of the present invention is that the albumen of SEQ.ID.NO.2 can be used as the hydrophobia adjuvant.By the albumen that recombinant expressed and SBP have 80% above homology, comprise with various associated forms such as the fusion of other antigenic components (as various protein polypeptides), polymerization, mixing and use the effective form of generation synergistic function together.

This albumen is to hydrophobia affinity result of experiment: with different dilution vaccine liquid (1:5,1:10,1:20,1:40,1:80) bag by 96 hole enzyme plates, it is anti-as one to hatch back adding SBP, anti-human IgG is that the two anti-ELISA that carry out react, the OD value that records contrasts with the result who does not add SBP, observes the affinity of SBP and hydrophobia.The result proves that SBP and rabies virus have extremely strong avidity, affinity costant Ka〉10 ⁷L/mol.

This albumen is to the influence of hydrophobia immune effect: laboratory animal is divided into test group and control group.With the mouse is example, uses the hydrophobia immune mouse according to a conventional method, subcutaneous injection.Got blood from mouse tail vein, separation of serum in the 12nd, 20,28,42 day after immunity.Measure the titre of anti-rabies virus antibody in the mouse body with the ELISA method.The difference of SPSS software analysis test group and control group.Found that this albumen can obviously strengthen the titre of mouse internal antibody, promptly this albumen can strengthen the immune effect of hydrophobia.

This shows; this albumen has extremely strong affinity to hydrophobia; utilize this albumen can strengthen the immunogenicity of hydrophobia; can reduce rabies virus antibodies and reach time of protective effect titre in vivo, help generating at short notice in the body rabies virus antibodies with protectiveness.Therefore can be adjuvant and be applied to the hydrophobia field with its synergy molecule, improve the effectiveness of existing hydrophobia, reduce rabic sickness rate and lethality rate as a kind of hydrophobia.

Description of drawings

This albumen of Fig. 1 is to the affinity of hydrophobia.

The influence that this albumen of Fig. 2 is tired to Balb/c mouse anti rabies virus antibodies.

Embodiment

One, the acquisition of gene order and transformation

Mode by synthetic, outer-gene amplification splicing is modified or same sense mutation the SBP gene order, obtains multiplely to have the gene order (SEQ.ID.NO.3-5) of identical function with SBP.For example, N or C end at this cDNA are introduced 6 * his purification tag and enteropeptidase restriction enzyme site DDDDK, 6 * his purification tag helps the affinity purification of purpose fusion rotein, and the enteropeptidase restriction enzyme site can make the fusion rotein of purifying cut away 6 * his tag and DDDDK under the effect of enteropeptidase.

According to SBP and the three-dimensional cooperating type of HBsAg, the analytical results of static distribution particularly, the method of employing point mutation and gene recombination is carried out structure of modification to SBP, and (as: V3D T8D), obtains to have the protein sequence (SEQ.ID.NO.8-9) of reinforced immunological synergistic function.

Two, gene engineering expression and purifying target protein

1, prokaryotic expression.Introduce 6 * his purification tag and enteropeptidase restriction enzyme site DDDDK at the N of this cDNA end.The dna clone that this is complete is in the pBV220 prokaryotic expression carrier, and transformed into escherichia coli screens positive transformant.The picking positive monoclonal is cultivated the trip temperature of going forward side by side and induced fermentation in liquid nutrient medium: at first at 37 C, 250rpm cultivates the intestinal bacteria of conversion to OD ₆₀₀=0.5 ~ 0.6, be warming up to 42 ℃ then rapidly and begin to induce fermentation 5 hours.Centrifugal collection thalline after the fermentation ends, the cracking thalline is to extract inclusion body, and the thick pure back of inclusion body finally obtains target protein with Ni-NTA agarose post affinity purification after the dialysis renaturation.The needed just target protein of albumen that is obtained with Western and ELISA experimental verification;

2, yeast eukaryotic expression.Said gene is cloned into carrier for expression of eukaryon pICZ α (a, b, c) transform pichia spp (GS115), carrying out methanol induction in the BMMY substratum cultivates, recombinant protein can separate purifying with ion exchange column etc. through the affinity column of the crosslinked SPA of having, ConA, mAb, has obtained the target protein of higher degree at last;

3, eukaryotic cell expression.With said gene sequence and improved sequence clone thereof in mammalian cell expression vectors such as pEGFP-N1, transfection CHO cell, in the RPMI-1640 perfect medium, cultivate in a large number, harvested cell and under the prerequisite of not damaging protein-active lysing cell, through Ni-NTA agarose post and through the crosslinked affinity column affinity purification that SPA, ConA, mAb are arranged, obtain highly active target protein.

Three, SBP albumen is to the evaluation that influences of hydrophobia effect

1, this albumen is to hydrophobia affinity result of experiment: with different dilution vaccine liquid (1:5,1:10,1:20,1:40,1:80) bag by 96 hole enzyme plates, it is anti-as one to hatch back adding SBP, anti-human IgG is that the two anti-ELISA that carry out react, the OD value that records contrasts with the result who does not add SBP, observes the affinity of SBP and hydrophobia.The result proves that SBP and rabies virus have extremely strong avidity, affinity costant Ka〉10 ⁷L/mol.

2, albumen is to the influence of hydrophobia immune effect: laboratory animal is divided into test group and control group.With the mouse is example, uses the hydrophobia immune mouse according to a conventional method, subcutaneous injection.Got blood from mouse tail vein, separation of serum in the 12nd, 20,28,42 day after immunity.Measure the titre of anti-rabies virus antibody in the mouse body with the ELISA method.The difference of SPSS software analysis test group and control group.

Result (Fig. 1, Fig. 2) finds that SBP and hydrophobia have extremely strong affinity interaction.Experiment mice has reached 8000 at back 14 days left and right sides antibody titers of immunity, and control group antibody after immune 14 days does not produce yet.In immunity back 20 days, the mouse antibodies of experimental group was tired and has been reached 10000, and the antibody of control group only is 4000.Above presentation of results SBP albumen can strengthen the immune effect of hydrophobia by strengthening the ability that raises anti-rabies virus antibody in the mouse body with the avidity of hydrophobia.

Four, the concrete application

Traditional hydrophobia often can not improve the titre of internal antibody at short notice, thereby can not be well provides enough antibody horizontal to resist infecting of virus in latent period at rabies virus.This hydrophobia synergistic protein provided by the invention can significantly improve the titre level of rabies virus antibodies in the mouse body in short duration, obviously strengthen the immune effect of hydrophobia, therefore, can use it for the exploitation of hydrophobia adjuvant.In the injection hydrophobia, inject a certain amount of this albumen, can strengthen the immunogenicity of hydrophobia, increase tiring of the interior anti-rabies virus antibody of body at short notice, thereby improve the immune efficient of vaccine, and reduce the consumption and the frequency injection of hydrophobia.

Claims

1. hydrophobia synergistic protein is characterized in that nucleotides sequence classifies SEQ.ID.NO.1 as.

2. the gene of hydrophobia synergistic protein according to claim 1 of encoding is characterized in that aminoacid sequence is SEQ.ID.N0.2.

3. albumen that has with the described hydrophobia synergistic protein of claim 1 said function is characterized in that nucleotides sequence classifies as shown in one of SEQ.ID.NO.3--SEQ.ID.NO.5.

4. the protein sequence through modifying is characterized in that aminoacid sequence is shown in SEQ.ID.NO.6 or the SEQ.ID.NO.7.

5. method of expressing hydrophobia synergistic protein as claimed in claim 1, it is characterized in that concrete steps are: gene order is cloned into prokaryotic expression carrier pBV220 transformed into escherichia coli, or carrier for expression of eukaryon pICZ α (a, b, c) transform pichia spp (GS115), or in the pEGFP-N1 mammalian cell expression vector, transfection CHO cell, through abduction delivering and purifying, obtain a series of highly purified target proteins.

According to claim 1 albumen as the application of hydrophobia adjuvant.

7. be of the application of the protein gene of SEQ.ID.NO.2 as aminoacid sequence as the hydrophobia adjuvant.