CN1280310C - Fusion protein with cold proventing and curing function and its encoding gene and use - Google Patents

Abstract

The invention discloses a fusion protein with the effect of preventing and treating influenza, its encoding gene and its application, and aims to provide the fusion protein with the effect of preventing and controlling the influenza, its encoding gene and a vaccine medicine with the protein as an active ingredient. The fusion protein with the effect of preventing and treating influenza provided by the present invention is SEQ ID No.: 1 in the sequence listing, and its coding sequence is SEQ ID No.: 2 in the sequence listing. The fusion protein with the effect of preventing and treating influenza can be used as a vaccine for preventing influenza virus.

Description

Fusion protein with anti-influenza effect, its coding gene and application

technical field

The invention relates to a fusion protein capable of preventing and treating influenza, its encoding gene and its application, in particular to a fusion protein capable of preventing and controlling influenza, its encoding gene and a vaccine with the protein as an active ingredient.

Background technique

Influenza is a common and multiple infectious disease caused by viruses, which poses a great threat to human beings, especially the elderly, children and weak people. At the same time, influenza outbreaks also have a serious impact on the entire society and economic development. Existing influenza vaccine has following three kinds: (1) influenza virus inactivated vaccine, this vaccine is widely used in adult at present, is to utilize the pathogen after the influenza virus inactivation to make. However, the vaccine strain needs to be replaced in time with the antigenic variation of the influenza virus strain, otherwise the immune effect will not be guaranteed or even invalid. The virus required for this type of influenza vaccine needs to be propagated and collected in chicken embryos. Once a large-scale influenza outbreak occurs, it is often difficult to guarantee the supply of qualified chicken embryos; moreover, the chicken embryo protein that cannot be completely removed in this type of vaccine can cause infection in some people. Emergence of allergic symptoms (Virus Research 2004, 103:173-176). (2) Live attenuated influenza virus vaccine, which has been widely used in humans since 1960, is also made from the attenuated pathogen of influenza virus. The vaccine strain also needs to be replaced in time with the antigenic variation of the influenza virus strain. At present, screening experiments for new vaccine strains are being carried out at home and abroad. (3) Influenza virus subunit vaccine, which is the influenza vaccine currently used by foreign children. It uses genetically recombined virus surface antigens to induce immune protection. Virus surface antigens also need to be replaced in time with the antigenic variation of influenza strains, otherwise The immune effect is not guaranteed, or even invalid.

The general characteristic of current commercialized influenza vaccines is that they cannot deal with the antigenic variation of influenza virus strains. Influenza viruses are highly variable, and antigenic mutations continue to occur. The above-mentioned influenza vaccine cannot deal with the variation of influenza virus. The fundamental reason is that the design of the above-mentioned influenza vaccine is basically based on a surface membrane protein HA (hemagglutinin) of influenza virus. A large number of foreign studies have shown that influenza virus as positive chain For RNA viruses, its RNA-dependent RNA polymerase (RNA dependent RNA polymerase) lacks the proof reading activity of eukaryotic DNA polymerase during genome replication. Therefore, the continuous mutation of the main surface protein antigen HA of influenza virus causes the immune escape of influenza virus, and the mutation rate of HA is 6.7×10 ^-3 (Annu Rev Genet 2002 36 305-332).

Since the 1990s, more studies have been transferred to another surface membrane protein M2 of influenza virus, and most of the research work is to use the entire extracellular domain sequence (aa1-24) of the M2 protein as the influenza virus. Design targets for viral vaccines. This is mainly because the M2 protein has outstanding conservation compared with HA, and a large number of previous studies have shown that the extracellular domain sequence of the M2 protein can induce a very effective protective humoral response (Nature Medicine 1999, 5: 1157-1163; Vaccine 2003, 21:2616-2626; Vaccine 1995, 13:1339-1342; Vaccine 2004, 22:2993-3003; Virus Research 2004, 103:173-176; Euro J Immunol 2002, 32:3305-14).

At present, most of the influenza vaccines designed based on the M2e epitope of influenza virus adopt the design idea of coupling the M2e epitope polypeptide to the carrier protein (Vaccine 2003, 21: 2616-2626; Vaccine 2004, 22: 2993-3003; Virus Research 2004, 103 : 173-176; Euro J Immunol 2002, 32: 3305-14), such an influenza vaccine design cannot guarantee the number of M2e epitope polypeptides successfully coupled to the carrier protein on the one hand; Some toxic chemical coupling agents are used in the carrier coupling process, such as the commonly used MBS (m-maleimidobenzoyl-N-hydroxy succinimid ester) and glutaraldehyde, as well as the DMSO that may be used in the process of melting polypeptides are all from Sigma Clearly indicate toxic substances, which will inevitably affect the production and promotion of this type of influenza vaccine.

A/PR/8/34 is one of the representative strains of human influenza A virus H1N1 subtype, usually spherical, with a diameter of 80-120nm. Negative-strand RNA virus, the genome length is 13.6Kb, and there are 10 clear protein products in the virus replication cycle. Passage and adapt the A/PR/8/34 virus in mice (Balb/c), so that the mice become ill and die, and the mouse lung-adapted strain of the mouse lethal type can be obtained, which is generally used for the death of mice of influenza virus at home and abroad Use strains for the type virus challenge model.

Contents of the invention

The purpose of the present invention is to provide a fusion protein and its coding gene which can prevent and treat influenza.

The fusion protein with anti-influenza effect provided by the present invention is named GST-(M2e) ₁₆ , which is the protein shown in SEQ ID No. 1 in the sequence listing:

SEQ ID No. 1 in the sequence listing consists of 645 amino acid residues. In sequence 1, amino acid residues from the 1st to 224th of the amino terminal are glutathione transferase, amino acid residues from the 225th to 226th of the amino terminal are connecting peptide GS, and amino acid residues from the 227th to 640th of the amino terminal The amino acid residues are 16 copies of the M2e protective epitope linearly connected together using a dipeptide linker (linker) RS, and the 641st-645th amino acid residue from the amino terminal is the telopeptide RSILL.

The gene encoding the above-mentioned fusion protein having the effect of preventing and treating influenza also belongs to the protection scope of the present invention.

The gene encoding the above-mentioned fusion protein having the effect of preventing and treating influenza can be the DNA sequence of SEQ ID No. 2 in the sequence listing.

SEQ ID No. 2 in the sequence listing consists of 1938 bases, and its coding sequence is from the 1st to the 1938th base at the 5' end.

Expression vectors, cell lines and engineering bacteria containing the genes of the present invention all belong to the protection scope of the present invention, such as pGEX-4T-M16, Escherichia coli BL21(DE3) containing pGEX-4T-M16.

The pair of primers for amplifying any fragment of the gene encoding the fusion protein having the effect of preventing and treating influenza is also within the protection scope of the present invention.

Another object of the present invention is to provide a vaccine against influenza virus.

The active ingredient of the vaccine for preventing influenza virus provided by the present invention is the above-mentioned fusion protein capable of preventing and controlling influenza.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above vaccine. The carrier includes conventional diluents, absorption enhancers and the like in the field of pharmacy.

The vaccine of the present invention can be made into injection, and the medicine in this dosage form can be prepared according to the conventional methods in the field of pharmacy.

The immunization dose of the above-mentioned vaccine is generally 0.5-2.5 mg fusion protein with the effect of preventing and treating influenza/kg body weight/time.

The fusion protein GST-(M2e) ₁₆ with the effect of preventing and treating influenza of the present invention is designed based on the protective sequence of the extracellular region of the influenza virus membrane protein M2, and is a combination of glutathione-S-transferase (Glutathione-S-transferase, GST) and a recombinant fusion protein of 16 copies of the M2e protective epitope linearly linked together using a dipeptide linker RS, specifically increased the copy number (epitope density) of the M2e protective epitope sequence. Moreover, GST-(M2e) ₁₆ is fermented by bioengineering method, the production process is mature, and its safety is greatly improved compared with chemically conjugated vaccines.

Experiments have shown that the humoral immune response against the M2e epitope sequence induced by GST-(M2e) ₁₆ was significantly stronger than that of the fusion containing only one or four copies of the M2e epitope sequence by immunizing mice or New Zealand white rabbits with the same immunization dose and route. Protein GST-M2e or GST-(M2e) ₄ induced M2e-specific humoral responses; and experiments on New Zealand white rabbits showed that GST-(M2e) ₁₆ was used as an immunizing agent to induce anti-serum M2e epitope sequence-specific antibodies The concentration was significantly higher than the antiserum induced by GST-M2e or GST-(M2e) ₄ as an immunizing agent; not only that, the antiserum induced by GST-(M2e) ₁₆ was significantly stronger than GST in recognizing the natural protein of influenza virus M2 -(M2e) or GST-(M2e) _4- induced antiserum; most importantly, the challenge experiment in mice showed that GST-(M2e) ₁₆ as an immunizing agent can 100% protect mice against lethal dose GST-(M2e) ₄ could only protect 50% of the mice from challenge respectively, while GST-M2e could not provide effective protection against the lethal influenza virus challenge. And a large amount of studies prove that the neutralizing epitope (the extracellular region amino acid sequence (aa1-24: MSLLTEVETPIRNEWGCRCNDSSD) of the influenza virus membrane protein M2 that the present invention uses is very common in all human influenza viruses. Conservation (Nature Medicine 1999,5:1157-1163; Vaccine 2004,22:2993-3003), so the GST-(M2e) ₁₆ of the present invention has potential protective ability against different subtypes of influenza virus, GST-(M2e ) ₁₆ will play an important role in the prevention and treatment of influenza.

Description of drawings

Figure 1A is the identification map of plasmid pGEX-4T-M1, pGEX-4T-M4, pGEX-4T-M16 by BamHI+XhoI double digestion

Figure 1B is the SDS-PAGE pattern of GST-M2e, GST-(M2e) ₄ , GST-(M2e) ₁₆

Fig. 2 is the immunoblotting pattern of the purified fusion protein GST-(M2e) ₁₆ , GST-(M2e) ₄ , GST-M2e interacting with the monoclonal antibody specific to the M2e epitope sequence

Fig. 3 is a legend description of immunoassays with GST, GST-M2e, GST-(M2e) ₄ , and GST-(M2e) ₁₆

Figure 4A-Figure 4C shows the M2e epitope specificity in the collected mouse serum 15 days after GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e were immunized for the first time, the second time and the third time respectively Determination curve of antibody titer

Fig. 4D-Fig. 4F are GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e 20 days after the first, second and third immunizations respectively, the M2e epitope specificity in the serum collected from New Zealand white rabbits Determination curve of antibody titer

Figure 5 shows the M2e epitope-specific antibodies in New Zealand white rabbit serum collected 20 days after the first, second and third immunizations using GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e respectively Concentration histogram of

6A-Figure 6B is the flow cytometry of PBS and New Zealand white rabbit serum collected 20 days after the third immunization with GST, GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e, respectively, to recognize the natural M2 protein of influenza virus Cytometer detection curve

Fig. 7 is the change curve of the body weight of the mice with the challenge time after the mice challenged to GST-(M2e) ₁₆ , GST-(M2e) ₄ or GST-M2e 15 days after the third immunization

Fig. 8 is the change curve of the survival rate of mice with the challenge time after the mice challenged 15 days after GST-(M2e) ₁₆ , GST-(M2e) ₄ or GST-M2e were immunized for the third time

Detailed ways

Example 1, Preparation and identification of GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e

1. Construction of three fusion protein genes: GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e

Oligonucleotide chains L1, L2, P1 and P2 were synthesized by Beijing Aoke Biotechnology Co., Ltd. using 8909 Expedite automatic nucleic acid synthesis instrument, and identified by gel purification. Their sequences are as follows: L1: 5`GCGC GGATCC ATGTCCCTGCTGACTGAAGTTGAAACTCCGATCCGTAACGAATG 3` (bases underlined are BamH I recognition sequences);

L2: 5` GTTATCA AGATCT GTCGGAGGAGTCGTTGCAACGGCAACCCCATTCGTTACGG 3` (the underlined base is the Bgl II recognition sequence);

P1: 5` GTGCGC GGATCC 3` (the underlined base is BamH I recognition sequence);

P2: 5` CTCGAG TTATCA AGATCT 3` (the underlined base at the 5' end is the Xho I recognition sequence, and the underlined base at the 3' end is the Bgl II recognition sequence)

Use P1 and P2 as upstream and downstream primers, use L1 and L2 as nested oligonucleotide chains for nested PCR (overlapping PCR), and use the following PCR (polymerase chain reaction) program to complete the artificial gene of the fusion protein Construct. The reaction system is: 2.5 pmol/L primers P1 and P2 4 μL each, 10 pmol/L nested oligonucleotide chains L1 and L2 each 5 μL as a template, 10 mmol/L dNTPs 1 μl, Taq enzyme 1U (purchased from Shanghai Shenneng Bocai Co., Ltd. Biological Company), Buffer 5 μl, made up to 50 μl with pure water. The reaction conditions were: denaturation at 94°C for 30 seconds, annealing at 45°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles, and finally extension at 72°C for 5 minutes. PCR products were detected by agarose gel electrophoresis and purified. The PCR product was mixed with the T-easy vector (purchased from Promega, USA) and ligated overnight at 4°C, and transformed into competent cells of Escherichia coli DH5α strain, and positive clones were screened by IPTG and X-gal blue-white spots. Extract the plasmids in the positive clones for sequencing, and name the ligation product containing the nested PCR product (with the nucleotide sequence of sequence 3 in the sequence listing) pGEM-T-MO, which contains the M2e epitope in the multiple cloning site Gene fragment, this gene fragment has the following characteristics: the 5' end of the fragment contains the recognition site of the restriction endonuclease BamHI; the 3' end contains the recognition site of the restriction endonuclease BglII and XhoI. BglII enzyme and XhoI (purchased from Takata Company) were used to double-digest pGEM-T-MO, and the vector fragment was purified and recovered using a DNA gel recovery kit (purchased from Shanghai Shenneng Bocai Co., Ltd.). Use T4 DNA ligase to connect a 614bp DNA fragment between the two restriction sites of the recovered double-enzyme-digested vector fragment (the DNA fragment is according to Liu Zuqiang, Chen Yinghua, Recombinant Technology: Design and construction of a recombinant epitope -peptide gene as a universalepitope-vaccine strategy, Journal of Immunological Methods 2004, 285:93-97, which has the nucleotide sequence of sequence 5 in the sequence table), the fragment has the following characteristics: the 5' end contains BglII (AGATCT) restriction endonuclease recognition site, followed by a stop codon (TAA), that is, 5'- AGATCT ATTCTACTG TAA 3'; the 3' end contains the XhoI recognition site; there is only a single BglII and XhoI, no BamHI site. The ligation product was transformed into competent cells of Escherichia coli DH5α strain, and positive clones were screened by IPTG and X-gal blue and white spots. Extract the plasmid sequencing in the positive clone, the ligation product containing the GST-M2e gene (with the nucleotide sequence of sequence 4 in the sequence listing) encoding an M2e epitope is named as pGEM-T-M1, and this gene encodes an M2e expression bit. Digest pGEM-T-M1 with BamHI+XhoI to recover small fragments, insert the vector fragment obtained after digesting pGEM-T-M1 with BglII+XhoI, transform the ligated product into competent cells of Escherichia coli DH5α strain, and use IPTG, X-gal Blue-white screening and sequencing yielded the ligation product pGEM-T-M2 containing the GST-(M2e) ₂ gene encoding two M2e epitopes. According to the above principle, basically repeat the above steps, digest pGEM-T-M2 with BamHI+XhoI to recover a small fragment, insert the vector fragment obtained after digesting pGEM-T-M2 with BglII+XhoI, and transform the ligated product into E. coli DH5α strain Competent cells were screened and sequenced by IPTG and X-gal blue-white spots, and the positive plasmid pGEM-T-M4 containing the GST-(M2e) 4 gene encoding ₄ M2e epitopes was obtained. Repeat the above steps twice according to the above principles to obtain the positive plasmid pGEM-T-M16, which contains the GST-(M2e) ₁₆ gene (with the nucleotide sequence of sequence 2 in the sequence listing) encoding sixteen M2e epitopes . In this way, three recombinant plasmids pGEM-T-M1, pGEM-T-M4 and pGEM-T-M16 were obtained, respectively having genes capable of encoding one, four and sixteen M2e epitopes. BamHI+XhoI digestion of the above three plasmids, recovery of the three epitope gene fragments released after digestion, respectively inserted into pGEX-4T-2 (purchased from Amersham Biosciences) digested by BamHI+XhoI, and positive clones pGEX were obtained after screening -4T-M1, pGEX-4T-M4, pGEX-4T-M16. These positive plasmids were identified by BamHI+XhoI double digestion (Figure 1A, lanes 1, 2, and 3 are GST-M2e gene, GST-(M2e) ₄ gene, GST-(M2e) ₁₆ gene) and DNA sequencing identification showed that the structure correct.

2. Expression and purification of GST-(M2e) ₁₆ , GST-(M2e) ₄ , and GST-M2e fusion proteins

The pGEX-4T-M1, pGEX-4T-M4, pGEX-4T-M16 and pGEX-4T-2 plasmids used as a negative control were transformed into BL21(DE3) bacteria by the CaCl ₂ method, and screened by IPTG and X-gal blue and white spots and sequencing to obtain positive clones. Each BL21(DE3) monoclonal strain transformed with a recombinant prokaryotic expression plasmid was subjected to the expression and purification of the fusion protein according to the following conditions. Shake culture in LB medium at 37°C and 220rpm overnight, and transfer to fresh LB medium at a ratio of 1:50 the next day to induce expression. The expression conditions are: when the bacteria grow to an OD A600 of about 0.6, add IPTG to a final concentration of 1 mmol/L, induce at 37°C for 3-4 hours (the longer the induction time, the higher the degradation rate of the target protein), and harvest the bacteria by centrifugation at 10,000rpm. Wash once with phosphate buffered saline (PBS, pH 7.2), and freeze the bacterial pellet at -20°C (freezing is conducive to the lysis of bacteria).

The fusion protein purification steps are as follows:

(1) Resuspend 500ml of bacterial pellet in 20ml of ice-cold PBS (pH7.2), add 20mg of lysozyme powder, add 2ml of 10% Triton X-100 PBS solution, add 100ul of 5mg/ml DNase and RNase, and stir rapidly for 30 minutes in an ice bath minutes until the solution is no longer viscous. At the same time, protease inhibitor PMSF was added to a final concentration of 100mmol/L.

(2) Ultrasonic lysis in an ice bath, and the supernatant was collected. The supernatant was filtered with a 0.45 μm filter membrane, and the filtrate was used for affinity purification of the fusion protein.

(3) The filtered supernatant was passed through an affinity column Glutathione-Sepharose 4B affinity column (purchased from Amersham Biosciences) at a flow rate of 30ml/h. Wash the column with 50-100ml 0.6-1M NaCl PBS (pH7.2) solution to remove most of the non-specifically adsorbed proteins.

(4) Under the monitoring of the FPLC system (Pharmacia), continue to equilibrate the affinity column with PBS (pH7.2), and use 10mM reduced glutathione 50mM Tris-HCl (pH8.0) solution to competitively elute the adsorbed recombinant Antigen, collect the eluted protein, and obtain the recombinant antigen for immunization.

Utilize 12% polyacrylamide gel electrophoresis (SDS-PAGE) to detect the purity of the fusion protein of purification, the result is as shown in Figure 1B, shows that obtains the fusion protein of electrophoresis pure (Fig. 1B, swimming lane 1,2,3 is GST respectively -M2e, GST-(M2e) ₄ , GST-(M2e) ₁₆ )

4. Protein concentration and antigenicity identification of GST-(M2e) ₁₆ , GST-(M2e) ₄ , and GST-M2e fusion proteins. Bradford method (Coomassie Brilliant Blue method) was used to measure the concentration of purified protein, wherein A630nm was measured and measured as The protein content is on the Y axis, and A630nm is the point marked with the standard solution on the X axis. The binomial method is used for curve fitting in Microsoft Excel, and then the concentration of the unknown protein is calculated using the obtained curve formula. The results showed that the protein concentrations of GST-(M2e) ₁₆ , GST-(M2e) ₄ , and GST-M2e expressed in step 2 were 0.30, 0.50, and 0.67 g/L, respectively.

The antigenicity of the fusion protein against the M2e epitope was determined by Immunoblotting (immunoblotting), and the specific steps were as follows:

(1) Firstly, 12% polyacrylamide gel electrophoresis (SDS-PAGE) of four samples GST-(M2e) ₁₆ , GST-(M2e) ₄ , GST-M2e and GST control was completed.

(2) Prepare 6 pieces of filter paper and 1 piece of mixed nitrocellulose filter membrane with the same size as the gel.

(3) Place three filter papers and nitrocellulose filter membranes on the anode plate of the electrotransfer device in turn, and soak them in the transfer buffer (the preparation of the transfer buffer: weigh 11.17g glycine, 2.42g Tris alkali, add water to 800ml, and use Adjust the pH to 8.0 with concentrated hydrochloric acid, and then add 200ml of methanol).

(4) Place the gel after electrophoresis on a nitrocellulose filter membrane covered with 3 pieces of filter paper and the cathode plate of the electrotransfer device. Add transfer buffer to the electrotransfer cell. Electrotransfer at constant voltage 14V for more than 8 hours.

(5) After the membrane transfer is completed, use 10×Ponceau staining solution (10×Ponceau staining solution formula: Ponceau S2g, trichloroacetic acid 30g, sulfosalicylic acid 30g, add water to 100ml) to mark the protein and cut tape. The excised nitrocellulose membrane was placed in PBS solution containing 1% skimmed milk powder and blocked for more than 2 hours at room temperature.

(6) The specific monoclonal antibody (5μg/ml) with M2e epitope diluted by PBS solution, the monoclonal antibody according to Liu Wanli, Zou Peng, Chen Yinghua, Monoclonal antibodies recognizing EVETPIRNepitope of influenza A virus M2 protein could protect mice from lethalinfluenza A virus challenge, Immunology letters 2004, 93: 131-136) reacted at room temperature for 30 minutes. Rinse the membrane three times with PBS solution, 10 minutes each time.

(7) Add a solution of secondary antibody (rabbit anti-mouse Ig-HRP covalently coupled to horseradish peroxidase (purchased from DAKO)) diluted in PBS at a concentration of 1 μg/ml and react in the dark for 30 minutes at room temperature. Rinse the membrane three times with PBS solution, 10 minutes each time.

(8) Add the freshly configured substrate solution and react in the dark. When the color of the protein band is observed to be obvious, remove the membrane from the substrate solution (substrate solution: 6mg DAB (3,3'-diaminobenzidine, Sigma) was dissolved in 10ml of PBS, 15 μl of 30% H ₂ O ₂ was added, mixed evenly and ready to use) was taken out and rinsed with deionized water to terminate the reaction, and photographed for preservation.

The results are shown in Figure 2, indicating that GST-(M2e) ₁₆ , GST-(M2e) ₄ and GST-M2e can all be recognized by M2e epitope-specific monoclonal antibodies, and have good immunogenicity. In Fig. 2, lanes 1, 2, 3 and 4 represent GST, GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ , respectively.

Example 2, GST-(M2e) _16- induced antibody level experiment

Figure 3 graphically shows the epitope density of M2e in GST, GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ with a legend, and uses M0, M1, M4 and M16 to represent GST, GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ .

Carry out the immunization experiment of mouse (BALB/c) and New Zealand white rabbit as animal model according to the following method:

A total of 32 female Balb/c mice weighing 16-20 grams were randomly divided into four groups, and 8 small mice were immunized intraperitoneally with immunogen (GST, GST-M2e, GST-(M2e) ₄ or GST-(M2e) ₁₆ ). mouse. For the first time, each mouse was injected with complete Freund's adjuvant (PIERCE company product) 1:1 emulsified recombinant immunogen diluted in PBS 50 μg, and the immune volume was 200 μl for the first subcutaneous multi-point (4 points) injection immunization, and each point was injected subcutaneously. 50 μL. Afterwards, they were immunized once every two weeks, and the last two immunizations were emulsified with incomplete Freund's adjuvant, and the immunization dose remained unchanged. Blood was drawn from the tail vein before immunization and 15 days after each immunization. Female New Zealand white rabbits weighing 1.7-1.9kg, three rabbits were subcutaneously immunized with each immunogen. Each rabbit was first injected with complete Freund's adjuvant (PIERCE company product) 1:1 emulsified recombinant immunogen diluted in PBS 200μg, the immune volume was 1000μl for the first subcutaneous multi-point (4 points) injection immunization, each point subcutaneously injected 250μL . Afterwards, they were immunized once every two weeks, and the last two immunizations were emulsified with incomplete Freund's adjuvant, and the immunization dose remained unchanged. Before immunization and 20 days after each immunization, blood was collected from the ear vein, and 20 days after the last immunization, a large amount of serum was obtained by bleeding from the carotid artery.

Recognition of serum for M2e epitope polypeptide identified by enzyme-linked immunosorbent assay (ELISA):

M2e epitope polypeptide (synthesized by U.S. Genemed Synthesis Company, San Francisco, CA, USA), its amino acid sequence is N-KSLLTEVETPIRNEWGCRCNDSSD-C, M2e polypeptide is coated on the polystyrene ELISA plate of solid phase (purchased from Costar) 15 days after each immunization, the antiserum obtained from blood collection was used as the first antibody to react with the antigen at room temperature for 1 hour; PBS-Tween (0.05% Tween, purchased from Sigma) was washed, and unbound antibodies were washed away; covalent coupling The rabbit anti-mouse Ig-HRP of horseradish peroxidase was used as the chromogenic secondary antibody (purchased from DAKO); after washing with PBS-Tween, the chromogenic substrate o-benzenediamine (OPD, purchased from Sigma) was added; finally A microplate reader (purchased from Bio-Rad, model 550) measured the absorbance at 450 nm. The results are shown in Figure 4A-Figure 4F, indicating that the antisera induced by GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ can recognize influenza virus M2 no matter in the mouse model or in the New Zealand white rabbit model M2e epitope polypeptide in the extracellular region of the protein. GST-(M2e) ₁₆ can induce a significantly enhanced M2e epitope peptide-specific immune response at the same immunization dose; 15 days after the initial immunization of mice (20 days in rabbits), GST-(M2e) ₁₆ The induced M2e-specific antibody titers exceeded 1:1600, while the fusion protein GST-(M2e) ₄ containing a lower M2e epitope density did not induce M2e-specific antibody titers exceeding 1 : 400 (FIG. 4A, FIG. 4D). 20 days after the second immunization of New Zealand white rabbits (15 days for mice), the M2e-specific antibody titer induced by GST-(M2e) ₁₆ exceeded 1:25600, while the M2e-specific antibody induced by the fusion protein GST-M2e The titers were only close to 1:1600 (Fig. 4B, Fig. 4E). After the third immunization, the M2e-specific antibody titer induced by GST-(M2e) ₁₆ in New Zealand white rabbits exceeded the M2e-specific antibody titer induced by GST-M2e more than ten times; in mice, GST- The level of M2e-specific antibody induced by (M2e) ₁₆ was also significantly higher than that induced by GST-M2e ( FIG. 4C , FIG. 4F ).

Example 3, GST-M2e, GST-(M2e) ₄ and GST-(M2e) _16- induced rabbit serum M2e-specific antibody concentration and the experiment of recognizing the natural M2 protein of influenza virus

1. Use the M2e epitope polypeptide-specific affinity chromatography column to identify the experimental affinity of GST-(M2e) ₁₆ , GST-(M2e) ₄ , and GST-M2e-induced rabbit antiserum for M2e epitope polypeptide-specific antibody concentration The principle and steps of the preparation of the chromatographic column: using the active group NHS on the column product Sepharose4-FAST of Pharmacia Company-easy to react with the NH ₂ -group of the M2e epitope polypeptide amino acid and relatively stable coupling The features together complete the preparation of the affinity chromatography column.

(1) Draw 1ml of NHS-activated_Sepharose4-FAST column material (Pharmacia company), and 2mg of M2e epitope polypeptide (synthesized by American Genemed Synthesis company, San Francisco, CA, USA; its amino acid sequence is N-KSLLTEVETPIRNEWGCRCNDSSD-C) Dissolve in deionized water. The NHS groups on the column were activated with 1 mM HCl.

(2) Put the column material in 1ml of 0.1M NaHCO ₃ (PH7.7), add 2mg of the dissolved M2e epitope polypeptide, shake gently for 0.5 hour, and then shake overnight on a shaker.

(3) Add 1ml of 0.5M Tris-0.5M NaCl (PH8.3) to the column after centrifugation, and block it for 2 hours to block unreacted NHS groups. Wash it several times with 20% ethanol for later use. .

Preliminary experiments have proved that the prepared M2e-specific affinity column can completely adsorb all M2e-specific antibodies in 1ml of rabbit serum, and the reuse efficiency of this affinity column is high.

The determination steps of M2e epitope polypeptide specific antibody concentration in rabbit serum are as follows:

(1) Use 5ml ultrapure water and 5ml PBS (pH7.2) to wash the M2e epitope peptide-specific affinity column that was originally stored in 20% ethanol.

(2) 1 ml of rabbit serum obtained from GST, GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ immunization was centrifuged at 12,000 g for 5 minutes at high speed, and diluted with 4 ml of PBS (pH 7.2) for 5 times, and filtered with a 0.45 μm membrane filter.

(3) The filtered serum was passed through the M2e-specific affinity column at a flow rate of 30ml/h (the column was passed in a 4°C refrigerator to minimize protein denaturation), and the sample was loaded repeatedly.

(4) Wash the column with 5-10ml PBS (pH7.2) solution to remove most of the non-specifically adsorbed proteins.

(5) Under the monitoring of the FPLC system (Pharmacia), continue to equilibrate the affinity column with PBS (pH 7.2), and use 0.15 mM glycine solution (pH 2.5) to elute the adsorbed M2e-specific antibody.

(6) Use FPLC system to monitor the elution peak of eluted M2e-specific antibody, and use FPLC system software to integrate to obtain the mass (μg) of M2e-specific antibody corresponding to this elution peak.

(7) Divide this mass by the initial 1 ml rabbit serum used for initial loading to obtain the specific antibody concentration (unit μg/ml) of the M2e epitope polypeptide in the rabbit serum.

The results are shown in Figure 5, indicating that GST-(M2e) ₁₆ induced relatively high levels of M2e in rabbit serum each time 20 days after the primary immunization, 20 days after the second immunization, and 20 days after the third immunization Epitope peptide-specific antibody concentration, and significantly higher than the fusion protein GST-(M2e) ₄ , the concentration of M2e-specific antibody induced by GST-M2e; in the serum collected 20 days after the third immunization The concentration is as high as 420μg/ml. In FIG. 5 , M0, M1, M4 and M16 represent GST, GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ , respectively.

2. Detection of the natural M2 protein of influenza virus in rabbit serum induced by influenza virus fusion protein vaccine by flow cytometry (FACS)

10 ⁷ -10 ⁸ MDCK cells (purchased from the American Type Culture Collection, ATCC) were inoculated with 10 ⁵ -10 ⁶ influenza virus A/wuhan/359/95 (A/wuhan/359/95 is human influenza A One of the representative strains of virus (H3N2 subtype), usually spherical, with a diameter of 80-120nm. Negative-strand RNA virus, the genome length is 13.6Kb, and there are 10 clear protein products in the virus replication cycle, see " Influenza Virus and Its Experimental Technology", Guo Yuanji, Cheng Xiaowen, China Three Gorges Publishing House), cultured at 34 degrees Celsius for 18 hours; then the MDCK cells (Nature Medicine 1999, 5 : 1157-1163), with PBS and 1:400 dilution antiserum 20 days after the third immunization of New Zealand white rabbits with GST, GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ at room temperature respectively Bath for 1 hour; use FITC-labeled goat anti-rabbit Ig-FITC (purchased from DAKO, Danmark) as the luminescent secondary antibody, and use FACSCalibur (purchased from BD Biosciences, Pharmingen) to detect the recognition situation. The results are shown in Figure 6A-Figure 6B, indicating that the antiserum (1:400) induced by the fusion protein with the M2e epitope after three immunizations can recognize the natural M2 protein of influenza virus (curves 3, 4, 5), where Antisera induced by GST-(M2e)16 (curve 5) were significantly more capable of recognizing influenza virus native M2 protein than those induced by fusion protein GST-(M2e) ₄ , GST-M2e (curves 4 and 3, respectively). In Fig. 6A-Fig. 6B, curve 1 is the recognition result of PBS and the natural M2 protein of influenza virus, curve 2 is the recognition result of the antiserum induced by GST control protein and natural M2 protein of influenza virus, and curves 3, 4 and 5 are respectively containing The recognition results of antisera induced by the fusion proteins of M2e epitope sequences GST-M2e, GST-(M2e) ₄ and GST-(M2e) ₁₆ and natural M2 protein of influenza virus.

Example 4, GST-(M2e) ₁₆ can 100% protect mice from the challenge of lethal dose of influenza virus

All four groups of mice (BALB/c) 15 days after the third immunization in Example 2 were given a lethal dose (about 10,000 live virions) of mouse lethal influenza virus (A/PR/8/34) (A/PR/8/34 is one of the representative strains of human influenza A virus (H1N1 subtype), usually spherical, with a diameter of 80-120nm. Negative-strand RNA virus, the genome length is 13.6Kb, in the virus There are 10 definite protein products in the replication cycle. The A/PR/8/34 virus is passed down and adapted in mice (Balb/c), and the mouse lung-adapted strain of the mouse lethal type can be obtained by making the mice morbidity and death, see "Influenza Virus and Its Experimental Technology", by Guo Yuanji, Cheng Xiaowen, China Three Gorges Publishing House) for virus attack. The body weight change of all mice, and the number of dead mice were recorded daily for 30 days after challenge. The results are shown in Figures 7 and 8. All mice immunized with the GST negative control group and the fusion protein GST-M2e containing only one M2e epitope lost weight significantly after challenge, and completely recovered within 10 days after challenge. death; the mice immunized with the fusion protein GST-(M2e) ₄ containing a lower M2e epitope density had a 50% survival rate after a lethal dose of challenge, but the body weight decreased significantly; while GST-(M2e) ₁₆ The immunized mice can not only resist the challenge of lethal dose of lethal influenza virus (A/PR/8/34) 100%, but also the body weight of the mice does not decrease significantly after the virus challenge. The mouse challenge experiment provides the most direct evidence for the effectiveness of GST-(M2e) _16. All the above experimental results show that the novel influenza virus fusion protein vaccine proposed by the present invention has good application value and will play a role in the prevention and treatment of influenza. important role.

Sequence Listing

<160>5

<210>1

<211>645

<212>PRT

<213> influenza virus belongs to human influenza virus (Orthomyxoviridae)

<400>1

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

210 215 220

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

225 230 235 240

Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Arg Ser Met Ser Leu Leu

245 250 255

Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn

260 265 270

Asp Ser Ser Asp Arg Ser Met Ser Leu Leu Thr Glu Val Glu Thr Pro

275 280 285

Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Arg Ser

290 295 300

Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly

305 310 315 320

Cys Arg Cys Ash Asp Ser Ser Asp Arg Ser Met Ser Leu Leu Thr Glu

325 330 335

Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser

340 345 350

Ser Asp Arg Ser Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg

355 360 365

Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Arg Ser Met Ser

370 375 380

Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg

385 390 395 400

Cys Ash Asp Ser Ser Asp Arg Ser Met Ser Leu Leu Thr Glu Val Glu

405 410 415

Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp

420 425 430

Arg Ser Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu

435 440 445

Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Arg Ser Met Ser Leu Leu

450 455 460

Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn

465 470 475 480

Asp Ser Ser Asp Arg Ser Met Ser Leu Leu Thr Glu Val Glu Thr Pro

485 490 495

Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Arg Ser

500 505 510

Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly

515 520 525

Cys Arg Cys Ash Asp Ser Ser Asp Arg Ser Met Ser Leu Leu Thr Glu

530 535 540

Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser

545 550 555 560

Ser Asp Arg Ser Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg

565 570 575

Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Arg Ser Met Ser

580 585 590

Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg

595 600 605

Cys Asn Asp Ser Ser Asp Arg Ser Met Ser Leu Leu Thr Glu Val Glu

610 615 620

Thr Pro Ile Arg Ash Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp

625 630 635 640

Arg Ser Ile Leu Leu

645

<210>2

<211>1938

<212>DNA

<213> influenza virus belongs to human influenza virus (Orthomyxoviridae)

<400>2

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatGa cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggttccgc gtggatccat gtccctgctg actgaagttg aaactccgat ccgtaacgaa 720

tggggttgcc gttgcaacga ctcctccgac agatccatgt ccctgctgac tgaagttgaa 780

actccgatcc gtaacgaatg gggttgccgt tgcaacgact cctccgacag atccatgtcc 840

ctgctgactg aagttgaaac tccgatccgt aacgaatggg gttgccgttg caacgactcc 900

tccgacagat ccatgtccct gctgactgaa gttgaaactc cgatccgtaa cgaatggggt 960

tgccgttgca acgactcctc cgacagatcc atgtccctgc tgactgaagt tgaaactccg 1020

atccgtaacg aatggggttg ccgttgcaac gactcctccg acagatccat gtccctgctg 1080

actgaagttg aaactccgat ccgtaacgaa tggggttgcc gttgcaacga ctcctccgac 1140

agatccatgt ccctgctgac tgaagttgaa actccgatcc gtaacgaatg gggttgccgt 1200

tgcaacgact cctccgacag atccatgtcc ctgctgactg aagttgaaac tccgatccgt 1260

aacgaatggg gttgccgttg caacgactcc tccgacagat ccatgtccct gctgactgaa 1320

gttgaaactc cgatccgtaa cgaatggggt tgccgttgca acgactcctc cgacagatcc 1380

atgtccctgc tgactgaagt tgaaactccg atccgtaacg aatggggttg ccgttgcaac 1440

gactcctccg acagatccat gtccctgctg actgaagttg aaactccgat ccgtaacgaa 1500

tggggttgcc gttgcaacga ctcctccgac agatccatgt ccctgctgac tgaagttgaa 1560

actccgatcc gtaacgaatg gggttgccgt tgcaacgact cctccgacag atccatgtcc 1620

ctgctgactg aagttgaaac tccgatccgt aacgaatggg gttgccgttg caacgactcc 1680

tccgacagat ccatgtccct gctgactgaa gttgaaactc cgatccgtaa cgaatggggt 1740

tgccgttgca acgactcctc cgacagatcc atgtccctgc tgactgaagt tgaaactccg 1800

atccgtaacg aatggggttg ccgttgcaac gactcctccg acagatccat gtccctgctg 1860

actgaagttg aaactccgat ccgtaacgaa tggggttgcc gttgcaacga ctcctccgac 1920

agatctattc tactgtaa 1938

<210>3

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

ggatccatgt ccctgctgac tgaagttgaa actccgatcc gtaacgaatg gggttgccgt 60

tgcaacgact cctccgacag atcttgataa ctcgag 96

<210>4

<211>768

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggttccgc gtggatccat gtccctgctg actgaagttg aaactccgat ccgtaacgaa 720

tggggttgcc gttgcaacga ctcctccgac agatctattc tactgtaa 768

<210>5

<211>614

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

agatctattc tactgtaagt tggggggcaa ctggacatgt gtgaaaggtg aaccagtggt 60

ctacacaggg gggcaagtaa aacaatgcaa atggtgtggc ttcgacttca acgagcctga 120

cggactcccca cactaccccca taggtaagtg cattttggca aatgagacag gttacagaat 180

agtagattca acggactgta acagagatgg cgttgtaatc agcgcagaag ggagtcatga 240

gtgcttgatc ggcaacacaa ctgtcaaggt gcatgcatca gatgaaagac tgggccctat 300

gccatgcaga cctaaagaga ttgtctctag tgcaggacct gtaaggaaaa cttcctgtac 360

attcaactac gcaaaaactt tgaagaacaa gtactatgag cccagggaca gctacttcca 420

gcaatatatg ctcaagggcg agtatcagta ctggtttgac ctggacgtga cagaccgcca 480

ctcagattac ttcgcagaat ttgttgtctt ggtggtggta gcactgttag gaggaagata 540

tgtcctgtgg ctgatagtga cctacatagt tctaagaatt ctgcagatat ccatcacact 600

ggcggccgct cgag 614

Claims (10)

1, having the fusion rotein of preventing and treating the influenza effect, is SEQ ID № in the sequence table: 1 amino acid residue sequence.

2, the described encoding gene of claim 1 with fusion rotein of preventing and treating the influenza effect.

3, gene according to claim 2 is characterized in that: described encoding gene with fusion rotein of preventing and treating the influenza effect is SEQ ID № in the sequence table: 2 dna sequence dna.

4, contain claim 2 or 3 described expression vectors with encoding gene of the fusion rotein of preventing and treating the influenza effect.

5, expression vector according to claim 4 is characterized in that: described expression vector is pGEX-4T-M16.

6, contain claim 2 or 3 described clones with encoding gene of the fusion rotein of preventing and treating the influenza effect.

7, contain claim 2 or 3 described engineering bacterias with encoding gene of the fusion rotein of preventing and treating the influenza effect.

8, engineering bacteria according to claim 7 is characterized in that: described engineering bacteria is the e. coli bl21 (DE3) that contains pGEX-4T-M16.

9, arbitrary segmental primer is right in amplification claim 2 or the 3 described encoding genes with fusion rotein of preventing and treating the influenza effect.

10, to be that claim 1 is described have a fusion rotein of preventing and treating the influenza effect for a kind of vaccine of flu-prevention virus, its activeconstituents.

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