CN104610456A - Preparation method and application of subunit vaccine for H7N9 subtype avian influenza - Google Patents

Abstract

The invention discloses a preparation method and application of an H7N9 subtype avian influenza subunit vaccine; the H7N9 subtype avian influenza subunit vaccine of the present invention is a fusion protein, and the fusion protein includes an H7N9 subtype avian influenza virus antigen HA1-2 protein and Salmonella typhimurium type I flagellin fliC with adjuvant effect. The fusion protein has the following amino acid sequence: (1) a protein consisting of the amino acid sequence shown in SEQ ID No.2; or (2) a homology with the amino acid sequence defined by SEQ ID No.2 of 80% to 100% % encoding the amino acid sequence of the same functional protein; or (3) a protein derived from (1) whose amino acid sequence shown in SEQ ID No. 2 is increased, deleted or replaced with one or more amino acids and has equivalent activity. The invention expresses the fusion protein in a short period and has good immunogenicity.

Description

A kind of preparation method and application of H7N9 subtype avian influenza subunit vaccine

technical field

The invention belongs to the field of immunology, and in particular relates to a preparation method and application of an H7N9 subtype avian influenza subunit vaccine.

Background technique

Influenza virus (Influenza Virus) is the causative agent of influenza, belonging to Orthomyxovoridae (Orthomyxovoridae) in classification, and influenza A virus genus. The viral genome is composed of 8 single-stranded negative-strand RNA fragments, each of which encodes a functional protein, namely PB1, PB2, PA, HA, NP, NA, M and NS genes. It is generally spherical, with a diameter of 80nm to 120nm, and has a capsule. There are mainly two kinds of glycoproteins on the surface of the capsule, namely, hemagglutinin (HA) and neuraminidase (Neuraminidase, NA). type and nine NA subtypes. The pathogenicity of influenza virus to the host is determined by multiple genes, among which the HA gene and its encoded protein are particularly important, which play a key role in the process of virus adsorption and transmembrane, and are important surface antigens, which stimulate the body to produce Neutralizing antibodies can neutralize viral infection.

Hemagglutinin is a glycoprotein containing 562-566 amino acids (Amino acid, aa), and its precursor molecule HA0 is cleaved into two parts, HA1 and HA2, by host proteases. Among them, HA1 contains 319-326 aa, and HA2 contains 221-222 aa. The globular region of the HA monomer is composed of most of HA1, and the stem region is mainly composed of HA2, and also contains a small part of HA1. Although HA1 is less conservative than HA2, after natural infection or vaccination, most of the protective neutralizing antibodies are directed against the globular region formed by HA1. In addition, X-ray crystallography results show that HA (57-264aa and 63-286aa) can Form a high-level structure close to the natural state.

At the end of February 2013, the H7N9 subtype avian influenza virus was first discovered in China, and then the epidemic developed rapidly. By October 2014, there were 453 laboratory-confirmed cases of human infection with the H7N9 subtype avian influenza virus, including 175 deaths. (http://www.who.int/). Gao et al. first reported that all gene fragments of the virus were derived from avian influenza virus and did not contain any human influenza virus gene fragments. The H7N9 subtype avian influenza virus is a new type of reassortant influenza virus with low pathogenicity in poultry. Influenza virus (LPAI), but its pathogenicity to humans is higher than that of H5N1, and it is difficult to detect and prevent, so it has attracted widespread attention.

Vaccine is one of the most effective ways to control virus infection, but the antigenicity of H7N9 subtype avian influenza virus is different from that of previous avian influenza viruses, so the original seasonal influenza vaccine and avian influenza vaccine lack immune protection against it At present, the influenza vaccines that have been approved for marketing in my country all adopt the traditional inactivation or cracking process, which has the disadvantages of long production cycle and low yield. Recombinant subunit vaccines, especially subunit vaccines expressed in Escherichia coli, have the characteristics of many protective antigenic epitopes, high expression efficiency, mature fermentation process, low production cost and easy large-scale production. Moreover, the subunit vaccine has high safety and is easy to be accepted by people. However, the main disadvantage of subunit vaccines is weak immunogenicity, and effective adjuvants are often needed to elicit ideal immune responses.

Flagellin is the main structural component of bacterial flagella and is a highly conserved protein. As a special inflammatory molecule, it has both antigenicity and adjuvant effect, and its adjuvant effect is mainly realized through the TLR5 signaling pathway. A large number of studies have shown that flagellin is a potential adjuvant, not only an inducer of innate immune response, but also helpful in inducing adaptive immune response.

At present, there is a lack of a preparation method and application of an H7N9 subtype avian influenza subunit vaccine with good immunogenicity.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method and application of an H7N9 subtype avian influenza subunit vaccine with good immunogenicity.

In order to achieve the above object, the present invention is achieved through the following technical scheme: the present invention provides a subunit vaccine of H7N9 subtype avian influenza, which is a fusion protein, and the fusion protein includes H7N9 subtype avian influenza virus antigen HA1-2 protein and Salmonella typhimurium type I flagellin fliC with adjuvant effect.

Further, the fusion protein has the following amino acid sequence:

(1) A protein consisting of the amino acid sequence shown in SEQ ID No.2; or

(2) An amino acid sequence that encodes the same functional protein with 80% to 100% homology to the amino acid sequence defined by the sequence SEQ ID No.2; or

(3) A protein derived from (1) that has the same activity as the amino acid sequence shown in SEQ ID No.2 by adding, deleting or replacing one or more amino acids.

A nucleic acid molecule of the present invention, which encodes the fusion protein.

Further, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No.1.

The present invention prepares the method for the subunit vaccine of described H7N9 subtype bird flu, comprises the following steps:

(1) overlap PCR amplifies the gene of the protein described in claim 1,

(2) connect to pCold carrier, obtain recombinant plasmid pCold-HA1-2-fliC,

(3) Transform the host strain E.coli BL21(DE3) to obtain the positive recombinant strain DE3(pCold-HA1-2-fliC),

(4) Fusion expression of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurium type I flagellin fliC in E. coli expression system; the target protein HA1-2-fliC was induced by IPTG, and Ni - Purify the target fusion protein with His tag by NAT affinity chromatography.

Further, in step (1), the acquisition of H7N9 subtype avian influenza virus HA1-2 gene: a. the extraction of H7N9 subtype avian influenza virus genomic RNA;

b. RNA reverse transcription to synthesize cDNA;

c. Cloning of HA1-2 gene of H7N9 subtype avian influenza virus; PCR amplification of fliC gene; overlap PCR amplification of HA1-2-fliC gene; recovery and purification of PCR products.

Further, in step (2), the PCR product HA1-2-fliC recovered by 1% agarose gel electrophoresis was connected to the prokaryotic expression vector pCold to obtain the recombinant plasmid pCold-HA1-2-fliC;

In step (3), the transformation of the connection product: the recombinant plasmid pCold-HA1-2-fliC, which transforms the host bacterium E.coli DH5a, obtains positive recombinant bacteria DH5a (pCold-HA1-2-fliC); Double digestion and sequencing identification;

Transformation of the ligation product: Transform the recombinant plasmid pCold-HA1-2-fliC into the host strain E.coli BL21(DE3) to obtain a positive recombinant strain DE3(pCold-HA1-2-fliC);

In step (4), expression of fusion protein HA1-2-FliC; purification of fusion protein HA1-2-FliC.

Further, in step (1), the cloning of H7N9 subtype avian influenza virus HA1-2 gene:

According to the nucleic acid sequence of H7N9 subtype avian influenza virus hemagglutinin HA in GenBank, two pairs of primers were designed, HA1-2-F'/HA1-2-R' and HA1-2-F/HA1-2-R;

Among them, the 5' end of HA1-2-F' and the 3' end of HA1-2-R' are respectively introduced with restriction enzyme sites SacI and HindIII, which are primers for amplifying a single HA1-2 gene; and primer HA1 -2-F and 5' end of HA1-2-R are respectively introduced restriction enzyme site EcoR I and flexible peptide (Gly ₄ Ser) ₃ coding gene; it is the primer used to amplify the HA1-2 gene in the fusion fragment ;

Primer HA1-2-F' has the nucleotide sequence of SEQID NO.3;

Primer HA1-2-R' has the nucleotide sequence of SEQID NO.4;

Primer HA1-2-F has the nucleotide sequence of SEQID NO.5;

Primer HA1-2-R has the nucleotide sequence of SEQID NO.6.

Further, in step (1), PCR amplification of the fliC gene: design primers fliC-F and fliC-R according to the sequence of the Salmonella typhimurium type I flagellin fliC gene, wherein 5 of the primers fliC-F and fliC-R The gene encoding the flexible peptide (Gly ₄ Ser) ₃ and the restriction enzyme site Xba I were respectively introduced into the 'end;

Primer fliC-F has the nucleotide sequence of SEQID NO.7;

Primer fliC-R has the nucleotide sequence of SEQID NO.8.

The application of the subunit vaccine of the present invention in the immune prevention of H7N9 subtype avian influenza.

Beneficial effects: the protective antigen of the highly immunogenic subunit vaccine prepared by the present invention is the HA1-2-fliC fusion protein, which has a short cycle of expressing the fusion protein, and the expression product has biological characteristics similar to natural products and good immunogenicity. In the present invention, the H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 gene is connected to the N-terminal of the Salmonella typhimurium flagellin gene through a flexible peptide, and the flagellin and the HA1-2 protein are fused and expressed through an Escherichia coli prokaryotic expression system, It overcomes the shortcomings of traditional inactivation or cracking process, long production cycle, low yield, and insufficient immunogenicity of subunit vaccines, so it is of great significance to the development of new H7N9 subtype avian influenza vaccines.

Description of drawings

Fig. 1 is the electrophoresis figure of target gene PCR amplification product;

Fig. 2 is the enzyme digestion identification diagram of recombinant plasmid pCold-HA1-2;

Fig. 3 is the enzyme digestion identification diagram of the recombinant plasmid pCold-HA1-2-fliC;

Fig. 4 is the SDS-PAGE result figure of recombinant bacterial DE3 (pCold-HA1-2) expression product;

Fig. 5 is the SDS-PAGE result figure of the expression product of recombinant bacteria DE3 (pCold-HA1-2-fliC);

Fig. 6 is the SDS-PAGE result figure of protein after purification;

Figure 7 is a Western blot detection diagram of proteins HA1-2 and HA1-2-fliC;

Figure 8 is a TLR5 biological activity detection diagram of the fusion protein HA1-2-fliC;

Fig. 9 is the detection diagram of IgG antibody specific to HA1-2 in mouse serum 12 days after the second immunization and the third immunization;

Fig. 10 is the detection figure of H7N9 subtype avian influenza virus hemagglutination test;

Figure 11 is a detection chart of the titer of hemagglutination inhibitory antibody in mouse serum after 12 days of three immunizations;

Figure 12 is a graph showing the dynamic changes of antibody levels in serum of mice after three immunizations.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

According to the technical solution of the present invention, the inventor provides the following specific application examples. It should be noted that the following examples are only illustrative, and the present invention is not limited to these examples.

Figure 1 is the electrophoresis diagram of the PCR amplification product of the target gene; among them, M1: DL2000 DNA marker, M2: λ-EcoT14digest DNA marker, 1: HA1-2 amplification product, 2: fliC amplification product, 3: HA1-2- fliC amplification product.

Figure 2 is the enzyme digestion identification diagram of the recombinant plasmid pCold-HA1-2; among them, M1: λ-EcoT14 digest DNA marker, M2: DL 2000 DNA marker, 1: pCold-HA1-2 enzyme digestion product.

Fig. 3 is the enzyme digestion identification map of recombinant plasmid pCold-HA1-2-fliC; among them, M: λ-EcoT14digest DNA marker, 1: pCold-HA1-2-fliC enzyme digestion product.

Figure 4 is the SDS-PAGE results of the expression product of the recombinant strain DE3 (pCold-HA1-2); wherein, M: protein marker, 1: empty vector, 2: not induced, 3: induced precipitate, 4: induced supernatant.

Figure 5 is the SDS-PAGE result of the expression product of the recombinant strain DE3 (pCold-HA1-2-fliC); among them, M: protein marker, 1: empty vector, 2: not induced, 3: induced precipitation, 4: induced supernatant .

Figure 6 is the SDS-PAGE results of the purified protein; wherein, M: protein marker, 1: HA1-2 protein, 2: HA1-2-fliC fusion protein.

Figure 7 is a Western blot detection diagram of proteins HA1-2 and HA1-2-fliC;

Figure 8 is a TLR5 biological activity detection diagram of the fusion protein HA1-2-fliC;

Fig. 9 is the detection diagram of IgG antibody specific to HA1-2 in mouse serum 12 days after the second immunization and the third immunization;

Fig. 10 is the detection figure of H7N9 subtype avian influenza virus hemagglutination test;

Figure 11 is a detection chart of the titer of hemagglutination inhibitory antibody in mouse serum after 12 days of three immunizations;

Figure 12 is a graph showing the dynamic changes of antibody levels in serum of mice after three immunizations.

The invention provides a subunit vaccine of H7N9 subtype avian influenza, which is a fusion protein comprising H7N9 subtype avian influenza virus antigen HA1-2 protein and Salmonella typhimurium type I flagella with adjuvant effect Protein fliC. The HA1-2 protein in the fusion protein is composed of amino acids 62-284 of the main protective antigen hemagglutinin of the H7N9 subtype avian influenza virus, and flagellin as an adjuvant enhances the immune response of the HA1-2 protein, with Stronger immunogenicity.

The fusion protein has the following amino acid sequence:

(1) A protein consisting of the amino acid sequence shown in SEQ ID No.2; or

(2) An amino acid sequence that encodes the same functional protein with 80% to 100% homology to the amino acid sequence defined by the sequence SEQ ID No.2; or

(3) A protein derived from (1) that has the same activity as the amino acid sequence shown in SEQ ID No.2 by adding, deleting or replacing one or more amino acids.

A nucleic acid molecule of the present invention, which encodes the fusion protein according to claim 1 or 2. The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No.1.

The present invention prepares the method for the subunit vaccine of described H7N9 subtype bird flu, comprises the following steps:

1. Construct a recombinant plasmid containing the HA1-2 gene of the hemagglutinin fragment of the H7N9 subtype avian influenza virus and the Salmonella typhimurium type I flagellin fliC gene. The specific steps are as follows:

1. Acquisition of H7N9 subtype avian influenza virus HA1-2 gene: (1) Extraction of H7N9 subtype avian influenza virus genomic RNA, (2) RNA reverse transcription to synthesize cDNA, (3) H7N9 subtype avian influenza virus HA1- 2 gene cloning;

2. PCR amplification of fliC gene;

3. Overlap PCR amplification of HA1-2-fliC gene;

4. Recovery and purification of PCR products;

5. Ligation reaction: The PCR products HA1-2 and HA1-2-fliC recovered by 1% agarose gel electrophoresis were respectively connected to the prokaryotic expression vector pCold to obtain recombinant plasmids pCold-HA1-2 and pCold-HA1-2 -fliC;

6. Transformation of the ligation product: Transform the recombinant plasmids pCold-HA1-2 and pCold-HA1-2-fliC into the host strain E.coliDH5a respectively to obtain positive recombinant strains DH5a (pCold-HA1-2) and DH5a (pCold-HA1 -2-fliC).

7. Double enzyme digestion and sequencing identification of recombinant plasmids

8. Transformation of the ligated product: transform the recombinant plasmids pCold-HA1-2 and pCold-HA1-2-fliC into the expression host strain E.coli BL21(DE3) respectively, and obtain positive recombinant strains DE3(pCold-HA1-2) and DE3 (pCold-HA1-2-fliC).

2. Fusion expression of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurium type I flagellin fliC in E. coli expression system:

1. Expression of protein HA1-2 and fusion protein HA1-2-FliC;

2. Purification of protein HA1-2 and fusion protein HA1-2-FliC;

3. Western blotting identification;

4. Detection of TLR5 biological activity.

3. Immunogenicity of fusion expression product of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurium type Ⅰ flagellin fliC:

18 C3H/HeJ female mice aged 6-8 weeks were randomly divided into 3 groups, 6 mice in each group, and the immunization route was intraperitoneal injection, and the immunization was carried out in three times, and the antibodies in the serum of mice in each immunized group were detected after immunization The level and dynamic changes of antibodies in the body over time, and the hemagglutination inhibitory titer of antibodies in serum are also detected.

Example 1

The present invention prepares the method for the subunit vaccine of described H7N9 subtype bird flu, comprises the following steps:

1. Construct a recombinant plasmid containing the HA1-2 gene of the hemagglutinin fragment of the H7N9 subtype avian influenza virus and the Salmonella typhimurium type I flagellin fliC gene. The specific steps are as follows:

1. Acquisition of HA1-2 gene of H7N9 subtype avian influenza virus

(1) Extraction of H7N9 subtype avian influenza virus genome RNA:

The Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University donated chicken embryo allantoic fluid containing inactivated H7N9 subtype avian influenza virus, and 500 μL was taken to extract RNA.

① added to Add 200 μL chloroform to the allantoic fluid of Reagent ( 1/5 of the volume of Reagent), fully oscillate, and wait for the emulsified solution to be milky, and let it stand at room temperature for 5 minutes;

②Centrifugation (12000g, 15min, 4°C);

③Carefully take out the centrifuge tube. At this time, the homogenate is divided into three layers (colorless supernatant, white protein layer in the middle, and colored lower organic phase), and the supernatant is transferred to another new centrifuge tube ( Do not suck out the white middle layer);

④ Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube up and down to mix thoroughly, and let stand at 15-30°C for 10 minutes; centrifuge (12000g, 10min, 4°C);

⑤Cleaning of RNA precipitation: Carefully discard the supernatant, slowly add 1mL of 75% ethanol along the wall of the centrifuge tube, gently wash the tube wall, centrifuge (12000g, 5min, 4°C), and discard the ethanol;

⑥ Dissolution of RNA: Dry the precipitate at room temperature until it becomes transparent, add an appropriate amount of RNase-free water to dissolve the precipitate, dissolve completely, and store at -70°C;

⑦ Take a small amount of RNA solution to test the concentration.

(2) RNA reverse transcription to synthesize cDNA

Prepare the RT reaction system (10 μL) according to the requirements of PrimeScript ^TM RT reagent kit as follows:

Note: 10 μL system RNA should not exceed 1 μg.

Water bath at 37°C for 15 minutes, and water bath at 85°C for 5s to inactivate reverse transcriptase.

(3) Cloning of H7N9 subtype avian influenza virus HA1-2 gene:

According to the influenza virus (A/Hangzhou/1/2013(H7N9)) hemagglutinin HA nucleic acid sequence in GenBank, two pairs of primers were designed, HA1-2-F'/HA1-2-R' and HA1-2-F/HA1 -2-R. The 5' end of HA1-2-F' and the 3' end of HA1-2-R' were respectively introduced with restriction enzyme cutting sites SacI and HindIII (underlined), which are used to amplify a single HA1-2 gene.

HA1-2-F': 5'-CCC GAGCTC AAAGGGAAAAGGACAGTTGACC-3' SEQ ID NO.3

HA1-2-R': 5'-CCC AAGCTT GGCATCAACCTGTACT-3' SEQ ID NO.4

The 5' ends of primers HA1-2-F and HA1-2-R were respectively introduced with restriction enzyme site EcoR I and flexible peptide (Gly ₄ Ser) ₃ encoding gene (underlined) for amplifying fusion fragments HA1-2 gene.

HA1-2-F: 5'-CCG GAATTC AAAGGGAAAAGGACAGTTGACC-3' SEQ ID NO.5

HA1-2-R: 5'- CACCTCCGCTTCCACCTCCACC GGCATCAACCTGTACT-3' SEQID NO.6

Amplify the HA1-2 gene PCR reaction system (20 μL) as follows:

The reaction system was mixed and centrifuged briefly, and placed on a PCR instrument for amplification. HA1-2 gene reaction conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 50 s, 59°C annealing for 50 s, 72°C extension for 50 s, 30 cycles, 72°C final extension for 10 min.

2. PCR amplification of fliC gene:

Primers fliC-F and fliC-R were designed according to the sequence of Salmonella typhimurium type Ⅰ flagellin fliC gene, and the 5′ ends of the primers fliC-F and fliC-R were introduced into the gene encoding flexible peptide (Gly ₄ Ser) ₃ and restriction Restriction site Xba I (underlined).

fliC-F: 5′- AGGTGGAAGCGGAGGTGGTGGAAGC ATGGCACAAGTCATTAATA-3′

SEQ ID NO.7

fliC-R: 5′-CCG TCTAGA TTAACGCAGTAAAGAGAGGACG-3′SEQ ID NO.8

Amplify the fliC gene PCR reaction system (20 μL) as follows:

The reaction system was mixed and centrifuged briefly, and placed on a PCR instrument for amplification. Reaction conditions of fliC gene: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 50 seconds, annealing at 56°C for 50 seconds, extension at 72°C for 2 minutes, 30 cycles, and final extension at 72°C for 10 minutes.

3. Overlap PCR amplification of HA1-2-fliC gene

Using the above PCR products (HA1-2 gene and fliC gene) as templates and HA1-2-F and fliC-R as primers, the target gene HA1-2-fliC was amplified by the overlapPCR method.

Amplify the HA1-2-fliC gene PCR reaction system (20 μL) as follows:

The reaction system was mixed and centrifuged briefly, and placed on a PCR instrument for amplification. HA1-2-fliC gene reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 50 s, annealing at 56°C for 50 s, extension at 72°C for 2 min and 30 s, a total of 30 cycles, and final extension at 72°C for 10 min.

Take 2 μL of each PCR product in the above steps and add an appropriate amount of Loading Buffer for 1% agarose gel electrophoresis. The results showed that specific bands appeared at about 669bp, 1488bp and 2187bp respectively after electrophoresis of the HA1-2, fliC and HA1-2-fliC gene products amplified by PCR, which was consistent with the expected results. The results are shown in Figure 1.

4. Recovery and purification of PCR products

After nucleic acid electrophoresis, use a clean blade to cut out the gel containing the target fragment DNA under ultraviolet light. Use the DNA gel recovery kit to recover the target gene, and operate the reference DNA recovery kit.

5. Ligation reaction

The PCR products HA1-2 and HA1-2-fliC recovered by 1% agarose gel electrophoresis were digested separately with the prokaryotic expression vector pCold; the recovered target fragments were ligated with the vector overnight at 16°C.

The ligation reaction system (10 μL) is as follows:

6. Transformation of Ligation Products

①Take a 1.5mL finger tube containing 100μL competent E.coli DH5a cells from -70℃, and melt in an ice bath.

② Add 10 μL of connection solution under sterile conditions, and place in an ice bath for 30 minutes, during which time vibration should not be avoided.

③Heat shock at 42°C for 90s without vibration.

④ Ice bath for 10 minutes.

⑤Add 890 μL of anti-antibiotic-free LB medium, and incubate at 37°C, 200 rpm for 1-2 hours.

⑥ Centrifuge (4000rpm, 5min).

⑦Reserve 200 μL of supernatant, discard the excess, mix well, and smear on LB agar plate containing ampicillin antibiotic.

⑧ Place the incubator upright at 37°C until there is no obvious liquid flow on the plate, and incubate it upside down overnight, about 16-20h,

7. Double enzyme digestion and sequencing identification of recombinant plasmids

(1) The recombinant bacteria were identified by extracting the plasmid and double enzyme digestion

The reaction system (20μL) is as follows:

After bathing in water at 37°C for 3 hours, agarose electrophoresis was performed for observation.

The results showed that the recombinant plasmid pCold-HA1-2 had a target fragment of about 669 bp and a vector fragment of 4407 bp after enzyme digestion, which was in line with the expected results. The results are shown in Figure 2. Similarly, after digestion of the recombinant plasmid pCold-HA1-2-fliC, a target fragment of about 2187bp and a vector fragment of 4407bp appeared, the results are shown in Figure 3.

(2) Recombinant plasmid sequencing and sequence analysis

The clones DH5a (pCold-HA1-2) and DH5a (pCold-HA1-2-fliC) identified as positive by double enzyme digestion were cultured overnight, and the recombinant plasmids were extracted and sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing.

8. Introduce the correct recombinant plasmid into E.coli BL21(DE3) competent cells, and name the correctly identified recombinant plasmids as DE3(pCold-HA1-2) and DE3(pCold-HA1-2-fliC) .

2. Fusion expression of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurium type I flagellin fliC in E. coli expression system

1. Expression of protein HA1-2 and fusion protein HA1-2-FliC

Pick the recombinant strains DE3(pCold-HA1-2) and DE3(pCold-HA1-2-fliC) in the ampicillin-resistant LB liquid medium, culture at 37°C with shaking until the OD ₆₀₀ is 0.6-0.8, 15°C After standing for 30 minutes, IPTG was added to a final concentration of 0.5 mM, and the protein expression was induced by shaking culture at 15°C for 24 hours. The induced product was ultrasonically lysed and centrifuged, and the lysed supernatant and precipitate of the induced product were analyzed by SDS-PAGE to determine the solubility of the protein. The results showed that there were obvious target protein bands at about 26kDa and 82kDa respectively, which were consistent with the expected protein size, indicating that the HA1-2 protein and HA1-2-fliC fusion protein were successfully expressed. The results are shown in Figure 4 and Figure 5 .

2. Purification of protein HA1-2 and fusion protein HA1-2-FliC

Continue to induce the expression of a large amount of recombinant bacteria (800mL) according to the method in 1, according to Novagen's According to the Purification Kit, purify the protein, and take part of the purified protein for SDS-PAGE identification. The results showed that there were single target protein bands at about 26kDa and 82kDa respectively, which were consistent with the expected protein size and high purity. The results are shown in Figure 6.

3.Western blotting identification

The purified fusion protein HA1-2-fliC and protein HA1-2 were subjected to SDS-PAGE electrophoresis, transferred to nitrocellulose membrane, blocked with 5% skimmed milk powder at room temperature for 2 hours, and tested with mouse anti-H7N9 subtype avian influenza Virus polyantiserum and anti-flagellin polyantiserum were used as primary antibodies, diluted at 1:500, incubated overnight at 4°C; the next day, HRP-labeled goat anti-mouse IgG was used as secondary antibody, diluted at 1:5000, incubated at room temperature for 2 hours before ECL /DAB color development. The results showed that both the proteins HA1-2 and HA1-2-fliC could react with mouse anti-H7N9 subtype avian influenza virus polyantiserum, and the fusion protein HA1-2-fliC could also react specifically with mouse anti-flagellin polyantisera. All the proteins have good immunoreactivity, and the results are shown in Figure 7. Figure 7 is the Western blot detection diagram of proteins HA1-2 and HA1-2-fliC.

4. Detection of TLR5 biological activity

5×10 ⁴ HEK293-mTLR5 cells (100 μL) per well were cultured overnight in a 96-well plate. The next day, HEK293-mTLR5 cells were stimulated with commercially available flagellin, HA1-2 protein, and HA1-2-fliC fusion protein at a concentration of 100 ng/mL, and the supernatant was collected after 5 hours, and IL-8 was detected with Human IL-8ELISA kit Secretion level, to evaluate the TLR5 activity of the target protein. The results showed that the fusion protein HA1-2-fliC could induce the cells to secrete a high level of IL-8 (2200pg/mL), while the HA1-2 protein stimulated group only secreted a very low level of IL-8 (100pg/mL). Prism 5.0 software analyzed the results, and the fusion protein HA1-2-fliC induced the level of IL-8 secreted by cells to be significantly higher than that of HA1-2 (P<0.001). The results are shown in FIG. 8 , which is a detection chart of the biological activity of the fusion protein HA1-2-fliC TLR5.

3. Immunogenicity of fusion expression product of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurium type Ⅰ flagellin fliC

1. Materials

Experimental animals: 18 C3H/HeJ female mice aged 6-8 weeks were purchased from Nanjing Institute of Biomedicine, Nanjing University.

Immune protein: Recombinant target proteins HA1-2 and HA1-2-fliC purified by His tag in previous experiments.

2. Mouse Immunization Protocol

The 18 mice were randomly divided into 3 groups, 6 in each group, and the immunization route was intraperitoneal injection, 200 μL/mouse was immunized three times, the first immunization was not performed for antibody detection, and after the second immunization, the third immunization was performed according to the results of antibody detection. The method and dose are the same as the first immunization, as shown in Table 1:

Table 1

3. Blood collection and serum separation of experimental mice

Twelve days after the second and third immunizations, blood was collected from the orbital veins of each immunization group. The blood was placed overnight at 4°C, centrifuged at 4,000 rpm/min for 15 minutes at 4°C, and then the upper transparent serum was carefully sucked out, and the aspirated serum was frozen at -20°C. , Separate serum for antibody detection and hemagglutination inhibition test.

4. Detection of HA1-2 specific IgG antibody in mouse serum 12 days after the second and third immunization

The steps of indirect ELISA detection are as follows:

① Coating: Dilute the GST-HA1-2 protein to 1.5 μg/mL with carbonate buffer the day before the test, add 100 μL to each well with a microsampler, and overnight at 4 °C;

②Washing: The next day, wash the microplate with PBST containing 0.05% Tween-20, wash 4 times in total, 5 minutes each time;

③Sealing: Add 200 μL of blocking solution (1% BSA in PBST) to each well with a microsampler, and seal in a water bath at 37°C for 2 hours;

④Washing: wash the plate with PBST, wash 4 times in total, 5min each time;

⑤ Primary antibody incubation: Dilute the serum to be tested with blocking solution to the working concentration (1:100 dilution), add 100 μL of the serum to be tested diluent to each well, and dilute to the appropriate concentration according to the 2-fold dilution method in turn, and act in a water bath at 37°C for 2 hours;

⑥ Washing: wash the plate with PBST, wash 5 times in total, 5 minutes each time;

⑦Secondary antibody incubation: Dilute the HRP-labeled goat anti-mouse antibody to the working concentration with blocking solution, IgG (1:10000 dilution), add 100 μL to each well with a micro-sampler, and react in a 37°C water bath for 1 hour;

⑧ Washing: wash the plate with PBST, wash 6 times in total, 5 min each time;

⑨Color development: Add TMB substrate buffer for color development, 100 μL/well, 37°C in the dark for 10 minutes; 2M H ₂ SO ₄ solution to stop the reaction, 50 μL/well;

⑩Reading: OD ₄₅₀ value read by ELISA reader

The results showed that the average level of HA1-2-specific IgG antibodies in the serum of mice in the HA1-2-fliC immunized group reached 12,800 after the second immunization for 12 days, and reached 48,640 after 12 days of the third immunization. After analysis by GraphPad Prism 5.0 software, HA1-2- The fliC immunization group was significantly higher than that of the HA1-2 immunization group (P<0.05). The results are shown in Figure 9, which is the detection chart of HA1-2-specific IgG antibody in mouse serum 12 days after the second and third immunizations.

5. Hemagglutination inhibition test of mouse serum 12 days after three immunizations

(1) Hemagglutination test

① Add 25 μL PBS to each well of the hemagglutination plate;

② Add the inactivated H7N9 subtype avian influenza virus to the first well, make 2-fold serial dilutions in turn, and set up positive control wells and negative control wells at the same time;

③ Add 25 μL of 1% chicken erythrocyte suspension to each well, oscillate on a horizontal shaker for 1-2 minutes to mix well, and let stand at 37°C for 30 minutes to judge the result.

Hemagglutination test results: the hemagglutination value of the inactivated H7N9 subtype avian influenza virus was 2 ⁸ , and the results are shown in FIG. 10 . Fig. 10 is the detection figure of H7N9 subtype avian influenza virus hemagglutination test;

(2) Hemagglutination inhibition test

① Prepare 4 units of virus liquid according to the results in (1): divide the hemagglutination value of inactivated H7N9 subtype avian influenza virus by 4 as the dilution of 4 units of virus liquid, and dilute with PBS;

② Add 25 μL of PBS to each well of the hemagglutination plate, add 25 μL of the serum to be tested in the first well, and make 2-fold serial dilutions in turn, and set up positive control wells and negative control wells at the same time;

③ Except for negative control wells, add 25 μL of 4 units of virus solution to each well; shake on a horizontal shaker for 1-2 minutes, then let stand at 37°C for 15 minutes;

④ Add 25 μL of 1% chicken erythrocyte suspension to each well, oscillate on a horizontal shaker for 1-2 minutes to mix well, and let stand at 37°C for 30 minutes to determine the result.

The results showed that 12 days after the third immunization, the average titer of hemagglutination-inhibiting antibody in the serum of mice in the HA1-2-fliC immunized group reached 2 ⁵ , which was significantly higher than that in the HA1-2 immunized group (P<0.01) by GraphPad Prism 5.0 software analysis ), the results are shown in Figure 11, and Figure 11 is a detection chart of the titer of the hemagglutination-inhibiting antibody in the mouse serum after 12 days of triple immunization.

6. Dynamic changes of antibodies in mouse serum after three immunizations

0 days after the third immunization (i.e. 12 days after the second immunization) to detect the antibody titer in the serum of the mice. After the third immunization, the orbital vein blood was collected from each immunized group every 12 days, and the dynamic changes of the antibody in the mouse serum after the third immunization were monitored. The results were as follows: It was shown that 12 days after the third immunization, the level of HA1-2 specific antibody in the serum of mice in the HA1-2-fliC immunized group reached the highest level (48640 on average), and then the antibody level showed a downward trend until 84 days after the third immunization. The level of HA1-2-specific antibody in the serum of mice in the -2-fliC immunized group was still around 10 ⁴ , which were significantly higher than those in the HA1-2 immunized group (P<0.05). Dynamic changes of antibody levels in serum of mice after treatment.

The protective antigen of the highly immunogenic subunit vaccine prepared by the present invention is the HA1-2-fliC fusion protein, which has a short cycle of expressing the fusion protein, and the expression product has biological characteristics similar to natural products and good immunogen sex. In the present invention, the H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 gene is connected to the N-terminal of the Salmonella typhimurium flagellin gene through a flexible peptide, and the flagellin and the HA1-2 protein are fused and expressed through an Escherichia coli prokaryotic expression system, It overcomes the shortcomings of traditional inactivation or cracking process, long production cycle, low yield, and insufficient immunogenicity of subunit vaccines, so it is of great significance to the development of new H7N9 subtype avian influenza vaccines.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have For various changes and improvements, the protection scope of the present invention is defined by the appended claims, description and their equivalents.

Claims (10)

1. a subunit vaccine for H7N9 subtype avian influenza, is characterized in that: it is fusion rotein, and described fusion rotein comprises H7N9 subtype avian influenza virus antigen HA1-2 albumen and has the Salmonella typhimurtum I type flagellin fliC of adjuvant effect.

2. the subunit vaccine of H7N9 subtype avian influenza according to claim 1, is characterized in that: described fusion rotein has following aminoacid sequence:

(1) protein be made up of the aminoacid sequence shown in SEQ ID No.2; Or

(2) amino acid sequence homology limited with sequence SEQ ID No.2 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or

(3) aminoacid sequence shown in SEQ ID No.2 has the albumen derivative by (1) of same isoreactivity through increasing, lacking or replace one or more amino acid.

3. a nucleic acid molecule, the fusion rotein of its coding described in claim 1 or 2.

4. nucleic acid molecule according to claim 3, its nucleotide sequence is as shown in SEQ ID No.1.

5. prepare the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 1, it is characterized in that comprising the following steps:

(1) gene of albumen described in overlap pcr amplification claim 1,

(2) be connected to pCold carrier, obtain recombinant plasmid pCold-HA1-2-fliC,

(3) transformed Host Strains E.coli BL21 (DE3), obtained positive recombinant bacterium DE3 (pCold-HA1-2-fliC),

(4) H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and the amalgamation and expression of Salmonella typhimurtum I type flagellin fliC in escherichia expression system; By IPTG abduction delivering target protein HA1-2-fliC, adopt Ni-NAT affinity chromatography purifying with the object fusion rotein of His label.

6. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 5, is characterized in that: in step (1), the acquisition of H7N9 subtype avian influenza virus HA1-2 gene: the extraction of a.H7N9 subtype avian influenza virus geneome RNA;

B.RNA reverse transcription synthesis cDNA;

The clone of c.H7N9 subtype avian influenza virus HA1-2 gene; The pcr amplification of fliC gene; The overlap pcr amplification of HA1-2-fliC gene; The retrieve and purification of PCR primer.

7. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 6, it is characterized in that: in step (2), by the PCR primer HA1-2-fliC reclaimed through 1% agarose gel electrophoresis, be connected with prokaryotic expression carrier pCold, obtain recombinant plasmid pCold-HA1-2-fliC;

In step (3), connect the conversion of product: by recombinant plasmid pCold-HA1-2-fliC, it transforms Host Strains E.coli DH5a, obtains positive recombinant bacterium DH5a (pCold-HA1-2-fliC); The double digestion of recombinant plasmid and order-checking qualification;

Connect the conversion of product: by recombinant plasmid pCold-HA1-2-fliC, it transforms expresses Host Strains E.coli BL21 (DE3), obtains positive recombinant bacterium DE3 (pCold-HA1-2-fliC);

In step (4), the expression of fusion rotein HA1-2-FliC; The purifying of fusion rotein HA1-2-FliC.

8. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 7, is characterized in that: in step (1), the clone of H7N9 subtype avian influenza virus HA1-2 gene:

According to the nucleotide sequence of H7N9 subtype avian influenza virus hemagglutinin HA in GenBank, design two pairs of primers, HA1-2-F '/HA1-2-R ' and HA1-2-F/HA1-2-R;

Wherein 5 ' the end of HA1-2-F ' and the 3 ' end of HA1-2-R ' introduce restriction enzyme site Sac I, Hind III respectively, are the primers for the independent HA1-2 gene that increases; And the 5 ' end of primer HA1-2-F and HA1-2-R introduces restriction enzyme site EcoR I and flexible peptide (Gly respectively ₄ser) ₃encoding gene; For the primer merging HA1-2 gene in fragment that increases;

Primer HA1-2-F ' has the nucleotide sequence of SEQID NO.3;

Primer HA1-2-R ' has the nucleotide sequence of SEQID NO.4;

Primer HA1-2-F has the nucleotide sequence of SEQID NO.5;

Primer HA1-2-R has the nucleotide sequence of SEQID NO.6.

9. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 8, it is characterized in that: in step (1), the pcr amplification of fliC gene: according to Salmonella typhimurtum I type flagellin fliC gene order design primer fliC-F and fliC-R, wherein 5 ' the end of primer fliC-F and fliC-R introduces flexible peptide (Gly respectively ₄ser) ₃encoding gene and restriction enzyme site Xba I;

Primer fliC-F has the nucleotide sequence of SEQID NO.7;

Primer fliC-R has the nucleotide sequence of SEQID NO.8.

10. the application of subunit vaccine in the immunoprophylaxis of H7N9 subtype avian influenza described in any one of claim 1 or 2.