Recombinant hemagglutinin proteins and uses thereof, methods of expression, subunit vaccines

Abstract

This application discloses a recombinant hemagglutinin protein, its uses, expression methods, and subunit vaccines, belonging to the field of biomedical technology. The technical solution is as follows: a recombinant hemagglutinin protein, obtained by removing the transmembrane and intracellular regions of the HA protein sequence of the H7N9-235 strain and fusing a T4-foldon sequence at the C-terminus; or by retaining the full-length sequence of the HA protein of the H7N9-235 strain. The amino acid sequence of the recombinant hemagglutinin protein is shown in SEQ ID NO.1 or SEQ ID NO.2. The application also discloses a recombinant hemagglutinin protein with good immunogenicity and medical prospects based on the H7N9 strain. Furthermore, when a subunit vaccine is prepared using the recombinant hemagglutinin protein with the transmembrane and intracellular regions removed, the immunogenicity after secondary immunization is significantly enhanced.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and more specifically, relates to a recombinant hemagglutinin protein and its uses, expression methods, and subunit vaccines. Background Technology

[0002] The H7N9 subtype of avian influenza virus is one of the avian influenza viruses that pose a significant threat to poultry farming and public health. Since its emergence, its continued transmission in poultry and the risk of cross-species infection have received widespread attention. With the virus constantly undergoing genetic mutations and antigenic drift, existing prevention and control measures still face considerable challenges.

[0003] Currently, the prevention and control of avian influenza still relies primarily on traditional inactivated vaccines. While these vaccines have played a role in practical applications, they still have several shortcomings, such as a relatively slow rate of strain updates, certain biosafety risks during production, and limited ability to induce cellular immunity. Therefore, developing novel avian influenza vaccines with higher safety, more defined antigenic composition, ease of large-scale production, and good immunogenicity is of great significance.

[0004] Hemagglutinin (HA) protein is the main glycoprotein on the surface of avian influenza viruses and a key antigen for inducing neutralizing antibodies and protective immune responses. The native conformation of HA protein is typically trimeric, and its conformational integrity is closely related to immunogenicity. Therefore, conformational optimization design around the HA protein is an important direction in subunit vaccine development.

[0005] In existing technologies, the expression and vaccine development of HA proteins mainly focus on the construction of truncated soluble proteins. However, different antigen conformations differ in expression characteristics, secretion capacity, conformational stability, and immunogenicity. There is still a lack of mature technical solutions for systematic comparison and screening of HA proteins derived from H7N9 strains, combined with eukaryotic stable expression systems. In particular, how to improve conformational stability and immunogenicity while ensuring antigen expression efficiency remains a technical problem that needs to be solved in this field.

[0006] Therefore, it is necessary to provide a recombinant HA candidate antigen suitable for H7N9 subtype avian influenza virus and its stable expression system, so as to provide a reliable technical basis for subsequent subunit vaccine development and immunization program optimization. Summary of the Invention

[0007] The main objective of this application is to provide a recombinant HA antigen designed based on the H7N9 subtype avian influenza virus and a stable expression system capable of expressing the above-mentioned recombinant antigen. To this end, this application has designed two pathways for the hemagglutinin of the H7N9 virus: retaining the full length or removing the intracellular and transmembrane regions, resulting in two recombinant hemagglutinin proteins. Furthermore, a cell line capable of stably expressing the above-mentioned proteins has been constructed. In addition, this application has further tested subunit vaccines prepared using the above-mentioned recombinant proteins. In the experiments, it was found that, on the one hand, the vaccine prepared with the recombinant hemagglutinin protein that retains the full-length sequence has better immunoprotective ability; on the other hand, the subunit vaccine prepared with the recombinant hemagglutinin protein that removes the intracellular and transmembrane regions shows a significant improvement in the immunogenicity after secondary immunization.

[0008] To achieve the above objectives, this application provides a recombinant hemagglutinin protein, which is obtained by removing the transmembrane and intracellular regions of the HA protein sequence of the H7N9-235 strain and fusing a T4-foldon sequence at the C-terminus.

[0009] Alternatively, the full-length sequence of the HA protein of the H7N9-235 strain can be retained, and the nucleotide sequence encoding the recombinant hemagglutinin protein is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] It should be noted that this application further tested subunit vaccines prepared using the above-mentioned recombinant proteins, and found that, on the one hand, vaccines prepared with recombinant hemagglutinin proteins that retain the full-length sequence have better immunoprotective capabilities, and on the other hand, subunit vaccines prepared with recombinant hemagglutinin proteins that remove the intracellular and transmembrane regions show a significant improvement in immunogenicity after secondary immunization.

[0011] Preferably, the recombinant hemagglutinin protein is obtained by retaining the full-length sequence of the HA protein of the H7N9-235 strain, and the gene sequence encoding the recombinant hemagglutinin protein is shown in SEQ ID NO.1.

[0012] Preferably, the recombinant hemagglutinin protein is obtained by removing the transmembrane and intracellular regions of the HA protein sequence of the H7N9-235 strain and fusing a T4-foldon sequence at the C-terminus, and the gene sequence encoding the recombinant hemagglutinin protein is shown in SEQ ID NO.2.

[0013] In addition, this application also discloses the use of the recombinant hemagglutinin protein as described above in the preparation of subunit vaccines.

[0014] In addition, this application also discloses a subunit vaccine using the aforementioned recombinant hemagglutinin protein as an antigen.

[0015] In addition, this application also discloses an expression method for preparing the above-mentioned recombinant hemagglutinin protein, wherein the nucleotide sequence encoding the recombinant hemagglutinin protein is cloned into a vector to obtain a recombinant plasmid, which is then transfected into cells and expressed and purified to obtain the recombinant hemagglutinin protein.

[0016] Preferably, the carrier is a pEE12.4 carrier;

[0017] The recombinant plasmid was transfected into CHO cells and expressed, then purified to obtain recombinant hemagglutinin protein.

[0018] One of the above-mentioned technical solutions in this application has at least one of the following advantages or beneficial effects:

[0019] 1. Based on the HA sequence of the H7N9-235 strain, this application constructs both the antigen HA-full, which retains the complete structure, and the soluble antigen HA-foldon, which is fused with T4-foldon. These can be used to systematically compare the effects of different conformational designs on expression characteristics and immune efficacy.

[0020] 2. This application utilizes the CHO cell expression system combined with the GS screening strategy to obtain a cell pool that can stably express recombinant HA protein, providing a stable foundation for the continuous preparation of candidate antigens and process scale-up.

[0021] 3. The antigen HA-full obtained in this application can induce good humoral and cellular immune responses under a single immunization, showing strong immunogenicity.

[0022] 4. Although the antigen HA-foldon obtained in this application has a relatively weak immune effect under single immunization conditions, the antibody response is significantly improved after booster immunization, indicating that it has the potential to further enhance the protective effect through optimization of the immunization program.

[0023] 5. This application forms a relatively complete technical route from candidate antigen screening, conformational optimization design, establishment of eukaryotic expression system to preliminary immunological evaluation, which can provide experimental basis and technical support for the development of H7N9 subunit vaccines.

[0024] In summary, this application provides a recombinant HA candidate antigen suitable for H7N9 subtype avian influenza virus and its stable expression technology, which has good application prospects in candidate antigen screening and immunization strategy optimization. Attached Figure Description

[0025] Figure 1 This is the plasmid map of the recombinant plasmid PEE12.4 in Example 1;

[0026] Figure 2 This is an electrophoretic identification image of the linearized enzyme digestion product of the recombinant plasmid in Example 2;

[0027] Figure 3 The results of Western blot analysis of HA protein expression in Example 3 are shown.

[0028] Figure 4 The results of serum HI titer after a single immunization of SPF chickens in Example 6;

[0029] Figure 5 The results show the survival rate of SPF chickens after challenge with the virus in Example 6;

[0030] Figure 6 The results of the detection of cytokine IFN-γ after immunization of SPF chickens in Example 7;

[0031] Figure 7 The results show the serum HI titer of SPF chickens after booster immunization in Example 8. Detailed Implementation

[0032] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] The following are the raw materials and their suppliers involved in the following examples and subsequent experiments:

[0034] The H7N9 subtype avian influenza virus, A / Chicken / Guangdong / E157 / 2017 (H7N9-E157), was provided by the National Avian Influenza Laboratory (Guangzhou), College of Veterinary Medicine, South China Agricultural University. The HA gene sequence of the H7N9 subtype avian influenza virus used in this application was derived from the H7N9-235 strain. The molecular cloning vector involved was the pEE12.4 expression vector, and the host cell was Chinese hamster ovary cells (CHO cells). This application used a glutamine synthase (GS) screening system to obtain a pool of CHO cells stably expressing the target protein.

[0035] Example 1: Construction of recombinant HA protein of avian influenza virus

[0036] Based on the HA gene coding sequence of avian influenza virus A / Chicken / Guangdong / 235 / 2019(H7N9)(H7N9-235), and using the HA gene sequence of the H7N9-235 strain as a template, two different forms of recombinant antigens were designed. One is the recombinant antigen HA-foldon. This antigen retains the main extracellular antigenic region of the HA protein, removing the transmembrane and intracellular domains to enhance protein secretion and expression. Simultaneously, a foldon sequence derived from T4 phage fibrin is fused to its C-terminus to promote the formation of a stable trimeric conformation of the target protein. If necessary, a purification tag can be further attached to the C-terminus of the antigen for subsequent protein detection and identification. The other is the recombinant antigen HA-full. This antigen retains the full-length sequence of the HA protein, including the signal peptide, extracellular region, transmembrane domain, and intracellular domain, to maintain its native conformational characteristics and membrane-anchored expression form as much as possible. Codon optimization was performed on the coding sequences of the two antigens according to CHO cell preferences to synthesize the HA-foldon and HA-full coding genes.

[0037] The amino acid sequence encoding the recombinant antigen HA-foldon is shown in SEQ ID NO.1;

[0038] The amino acid sequence encoding the recombinant antigen HA-full is shown in SEQ ID NO.2;

[0039] The recombinant HA sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into the PEE12.4 vector, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0040] After the synthesized DNA sequence was verified by sequencing alignment, plasmids were extracted using a plasmid extraction kit (purchased from Tiangen Biotech Co., Ltd., Cat.#DP103-03), and named PEE12.4-HA-foldon and PEE12.4-HA-full. The plasmid map is shown below. Figure 1 (As shown in Table 1). The recombinant plasmid was double-digested with FastDigest Hind III and FastDigest EcoR I restriction endonucleases, as shown in Table 1. The digestion system was incubated in a 37°C water bath for 2 hours. After incubation, the digestion products were separated by 1% agarose gel electrophoresis at a constant voltage of 150V for 25 minutes. The results were recorded using a gel imaging system after electrophoresis. Figure 2 .

[0041] Table 1: Double Enzyme Digestion Reaction System

[0042] reagents volume 10×FastDigestingBuffer 5μL Recombinant plasmid DNA 1μg Hind Ⅲ 1μL EcoR Ⅰ 1μL Ultrapure water up to 50μL

[0043] The plasmid concentration was measured using a NanoPhotometer® N50 ultraviolet-visible spectrophotometer and stored at -20°C for later use.

[0044] Example 2: Transfection of CHO cells with recombinant plasmid

[0045] 1. Linearization of plasmids PEE12.4-HA-foldon and PEE12.4-HA-full

[0046] The plasmid obtained in Example 1 was digested with FastDigest Pvu I restriction endonuclease (purchased from Thermo Fisher Scientific, catalog number FD0624). The reaction system is shown in Table 2 below:

[0047] Table 2: Reaction of restriction endonuclease FastDigest Pvu I digestion system

[0048] Components volume plasmid 5μg FastDigest Pvu I enzyme 5μL 10×FastDigest Green Buffer 5μL RNase-free ddH2O Up to 50μL

[0049] After gentle mixing, incubate briefly. Incubate at 37°C for 30 min, then at 65°C for 5 min to inactivate the endonuclease. The linearized plasmid is then recovered by ethanol precipitation.

[0050] 1. Recombinant plasmid transfection of CHO cells

[0051] Select viable CHO cells in the logarithmic growth phase for transfection. Centrifuge an appropriate amount of cells at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 1 mL of cell culture medium to achieve a cell density of 50 × 10⁶ cells / mL. 6 Cells / mL; The linearized recombinant plasmid and cell suspension were mixed at a ratio of 10 μg: 1 mL, gently pipetted to mix, and incubated at room temperature for 20 min. After incubation, 1 mL of plasmid-cell mixture was transferred to two electroporation cuvettes, 500 μL each, and placed on ice for equilibration for 10 min. The parameters were set according to the electroporator's operating manual: voltage 300 V, pulse time 15 ms, single pulse. After wiping the condensate off the outer wall of the electroporator cuvette with sterile filter paper, the cuvette was inserted into the sample holder of the electroporator, and electroporation was performed strictly according to the instrument's operating procedures. After electroporation, the plasmid-cell mixture in the cuvette was quickly transferred to 30 mL of fresh CHO-012A medium (excluding L-Gln) and cultured at 37°C, 5% CO2, 120 rpm in a shaker incubator for 24 h.

[0052] 2. Screening of transfected cells

[0053] Cells cultured for 24 hours were transferred to sterile centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 30 mL of CHO-012A medium containing 50 μmol / L MSX. The pellet was then transferred to 125 mL sterile Erlenmeyer flasks and cultured at 37°C, 5% CO2, and 120 rpm in a shaker. From the start of the selection culture, cell density and viability were measured every 48 hours. Culture was continued for approximately 16 days until the cell density reached 4.0 × 10⁶ cells / mL. 6 The screening is considered complete only if the cell count / mL is greater than 90%.

[0054] 3. Expression of recombinant proteins

[0055] The selected cells were transferred to 500mL sterile Erlenmeyer flasks and placed in a 37℃, 5% CO2, 120rpm shaker incubator for protein expression culture. Samples were harvested after about 14 days.

[0056] Example 3: Preliminary identification of HA protein expression

[0057] 1. Protein sample preparation

[0058] Transfer the harvested sample to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min. Collect the supernatant after centrifugation to obtain the target protein. For cell pellet, resuspend in an equal volume of PBS buffer and mix well. Then, sonicate the cell pellet suspension according to the instructions of the sonicator for approximately 5 min. After sonication, centrifuge at 800g for 5 min and collect the supernatant. Pipette an appropriate amount of protein into a 1.5 mL EP tube and add protein loading buffer. Incubate in a boiling water bath for 10 min.

[0059] 2. Western blot identification of HA protein expression

[0060] First, a 10% protein gel was prepared for SDS-PAGE electrophoresis under constant voltage of 80V for 20 min, followed by constant voltage of 120V for 70 min. The membrane was then transferred under constant current of 200mA for 80 min. After transfer, the membrane was blocked with 5% skim milk at room temperature for 2 h, and washed three times with PBS for 5 min each time. His monoclonal antibody was diluted 1:7000 and incubated at room temperature for 1 h. The membrane was washed three times with PBST for 10 min each time, and finally washed once with PBS for 10 min each time. Horseradish enzyme-labeled goat anti-mouse IgG was diluted 1:10,000 and incubated at 4°C in the dark for 1 h. The membrane was washed three times with PBST for 10 min each time, and finally washed once with PBS for 10 min each time (horseradish enzyme-labeled goat anti-mouse IgG was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.). Finally, the NC membrane was scanned and analyzed using a dual-color laser imaging system to observe the specific target protein bands and photograph and save the images.

[0061] The results are as follows Figure 3 As shown, compared with the control group, a distinct band of approximately 75 kDa appeared in the supernatant lanes of the experimental group transfected with recombinant HA-foldon. The results indicate that CHO cells can successfully express recombinant HA protein, and HA-foldon can be secreted into the cell culture supernatant, with an increased expression level compared to the original sequence.

[0062] Example 4: Purification of Recombinant Protein

[0063] For the recombinant protein HA-foldon obtained in Example 2, the culture medium supernatant was filtered through a 0.45 μm filter membrane to obtain a clear supernatant; the supernatant was loaded into His Trap (Ni) pre-equilibrated with binding buffer (50 mM Tris-HCl, pH 7.8-8.0, 150-500 mM NaCl, 10-25 mM imidazole). 2+ -IMAC affinity chromatography column: Wash the column with wash buffer containing 10-100 mM imidazole to remove non-specifically bound contaminating proteins, then elute the bound target protein with wash buffer containing 200-500 mM imidazole; collect the eluted fractions and replace the buffer by dialysis to remove imidazole.

[0064] Example 5: Preparation of Genetically Engineered Subunit Vaccines

[0065] The recombinant proteins obtained in Examples 2 and 4 were used to prepare subunit vaccines in conjunction with SEPPIC MONTANIDE™ ISA 78VG adjuvant. According to the adjuvant instructions, an appropriate amount of recombinant HA protein sample was taken as the antigen solution and mixed with the adjuvant, then emulsified into a milky white, opaque, water-in-oil emulsion. This was then bottled in sterile vials, labeled with the subunit vaccine name, batch number, concentration, preparation date, etc., and stored at 4°C for later use.

[0066] Example 6: Efficacy evaluation of genetically engineered subunit vaccine in SPF chickens

[0067] 1. Experimental grouping and immunization

[0068] To verify the immunization efficacy of the genetically engineered subunit vaccine, an animal experiment was conducted in SPF chickens. Forty 14-day-old SPF chickens were randomly divided into four groups. Group 1 (n=10) was administered 10 μg of HA-foldon + 78VG vaccine; Group 2 (n=10) was administered 10 μg of HA-full + 78VG vaccine; Group 3 (n=10) was administered a laboratory-developed inactivated H7N9-E157 vaccine IV-E157 + 78VG; and Group 4 (n=10) served as a blank control using PBS buffer.

[0069] 2. Detection of antibody levels and challenge protection rate after immunization

[0070] Blood samples were collected at weeks 2 and 3 post-immunization for HI test to analyze specific antibody levels. In week 3, the avian influenza virus A / Chicken / Guangdong / E157 / 2017 (H7N9-E157) homologous strain was used at 10... 6 EID 50 The SPF chickens in each experiment were challenged with 0.2 mL of the vaccine via nasal drops. The status of the SPF chickens was recorded daily for 14 days post-challenge, and the vaccine's protection rate was analyzed at the end. On day 5 post-challenge, throat and anal swabs were collected from each group of SPF chickens to detect viral shedding.

[0071] 2.1 HI antibody level detection

[0072] The results are as follows Figure 4As shown, at week 2 post-immunization, the average HI antibody titer against the H7N9-157 strain was 6.2 log2 in the recombinant protein HA-full immunization group, 1.25 log2 in the HA-foldon immunization group, and 1.69 log2 in the inactivated vaccine immunization group. At week 3 post-immunization, the average HI antibody titer was 8 log2 in the recombinant protein HA-full immunization group, 2.75 log2 in the HA-foldon immunization group, and 4.38 log2 in the inactivated vaccine immunization group. No specific HI antibodies were detected in the PBS group throughout the entire experimental period.

[0073] 2.2 Survival rate and virus shedding of chickens after challenge

[0074] Chicken survival rate results as follows Figure 5 As shown, all SPF chickens in the PBS group died within 3 days after challenge. The survival rate of SPF chickens in the HA-full group was 100%, and no viral shedding was detected on day 5 after challenge, indicating a 100% inhibition rate of viral shedding. The survival rate of SPF chickens in the HA-foldon group was 100%, and viral shedding was detected in 8 chickens on day 5 after challenge, indicating a 20% inhibition rate of viral shedding (2 / 10). The survival rate of SPF chickens in the inactivated vaccine group was 100%, and no viral shedding was detected on day 5 after challenge, indicating a 100% inhibition rate of viral shedding.

[0075] Example 7: Detection of SPF Chicken Cell Immunological Response to Genetically Engineered Subunit Vaccine

[0076] 1. Experimental grouping and immunization

[0077] To verify the cellular immunization effect of the recombinant protein HA-foldon vaccine, 40 14-day-old SPF chickens were randomly divided into 4 groups. Group 1 (n=10) was administered 10 μg of HA-foldon + 78VG vaccine, Group 2 (n=10) was administered 10 μg of HA-full + 78VG vaccine, Group 3 (n=10) was administered the laboratory-inactivated H7N9-E157 vaccine IV-E157 + 78VG, and Group 4 (n=10) was administered PBS buffer as a blank control.

[0078] 2. Peripheral lymphocyte isolation and ELISPOT staining

[0079] Three weeks post-immunization, 2 mL of anticoagulated peripheral blood was collected from chickens. Peripheral blood lymphocytes were isolated using a chicken peripheral blood lymphocyte isolation kit and erythrocyte lysis buffer purchased from Beijing Solarbio Science & Technology Co., Ltd. The cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6Cells were seeded at 1 / mL onto ELISPOT plates (purchased from Shenzhen Dakowei Biotechnology Co., Ltd.) and 2 μg of stimulant was added. Cells were incubated at 37℃ with 5% CO2 for 18 h. The culture medium was discarded, and the cells were washed 5 times with PBS. Cells were incubated with 1 μg / mL MT7C10-biotin at room temperature for 2 h. The plates were then cleaned and washed 5 times with PBS. Streptomycin-alkaline phosphatase diluted 1:1000 was added, and the plates were incubated at room temperature for 1 h. The plates were then cleaned and washed 5 times with PBS. BCIP / NBT-plus was added for color development until distinct spots appeared. Cells were washed thoroughly with tap water to stop the color development and then air-dried. The results were analyzed and recorded using an ELISA spot imager.

[0080] The results are as follows Figure 6 The mean spot-forming units (SFU / 10) of the HA-full immunization group shown were 652.75 SFU / 10. 6 PBMCs; the mean SFU in the inactivated vaccine group and the PBS control group were 219 SFU / 10 6 PBMCs and 140.67 SFU / 10 6 PBMCs. This indicates that the HA-full immunization group can enhance the antiviral capacity of chickens and effectively activate the cellular immune response in SPF chickens.

[0081] Example 8: Efficacy evaluation of booster immunization of genetically engineered subunit vaccines in SPF chickens

[0082] 1. Experimental grouping and immunity

[0083] To verify the immunization efficacy of the genetically engineered subunit vaccine, an animal experiment was conducted in SPF chickens. Twenty 14-day-old SPF chickens were randomly divided into four groups. Group 1 (n=10) was injected with 10 μg of HA-foldon + 78VG vaccine, while Group 2 (n=10) served as a blank control with PBS buffer. A booster immunization with the same dose was administered three weeks after the initial immunization. Blood samples were collected at two and three weeks post-boost immunization for the Hit Chronic Antibody (HI) test to analyze specific antibody levels.

[0084] 2. Post-immunization antibody level detection

[0085] The levels of HI antibodies in the serum of each group were measured at 2 and 3 weeks after booster immunization. Results Figure 7 The mean HI antibody titer in the HA-foldon group increased from 5.67 log2 to 7.67 log2; no HI antibody was produced in the PBS control group throughout the experiment.

[0086] Results analysis:

[0087] 1. This application successfully constructed two H7N9 recombinant HA candidate antigens, HA-foldon and HA-full, and obtained a CHO cell pool that stably expresses the corresponding antigens. This demonstrates that the CHO expression system and GS screening scheme established in this application are feasible and can provide technical support for the stable preparation of recombinant HA antigens.

[0088] 2. The HA-full antigen provided in this application can induce good humoral and cellular immune responses under a single immunization, indicating that preserving the complete HA structure is beneficial to maintaining the native conformation of the antigen and enhancing immunogenicity.

[0089] 3. Although the HA-foldon antigen provided in this application has a relatively weak immune effect under single immunization conditions, the antibody response is significantly improved after booster immunization, indicating that it has the potential to further enhance the immune effect through optimization of the immunization program.

[0090] 4. As can be seen from the comparison between HA-full and HA-foldon, the characteristic of this application in antigen design is that candidate antigens are constructed from two directions: the natural full-length configuration and the soluble trimer configuration, thereby achieving systematic screening of the immune effects of HA antigens in different conformations.

[0091] 5. This application provides experimental basis for screening candidate antigens for H7N9 subunit vaccines, optimizing the design of HA protein conformation, establishing a CHO eukaryotic expression system, and optimizing immunization procedures, and has good application prospects.

Claims

1. A recombinant hemagglutinin protein, characterized in that, The recombinant hemagglutinin protein was obtained by removing the transmembrane and intracellular regions of the HA protein sequence of the H7N9-235 strain and fusing a T4-foldon sequence at the C-terminus. Alternatively, the full-length sequence of the HA protein of the H7N9-235 strain can be retained, and the nucleotide sequence encoding the recombinant hemagglutinin protein is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The recombinant hemagglutinin protein according to claim 1, characterized in that, The recombinant hemagglutinin protein was obtained by retaining the full-length sequence of the HA protein of the H7N9-235 strain, and the gene sequence encoding the recombinant hemagglutinin protein is shown in SEQ ID NO.

1.

3. The recombinant hemagglutinin protein according to claim 1, characterized in that, The recombinant hemagglutinin protein was obtained by removing the transmembrane and intracellular regions of the HA protein sequence of the H7N9-235 strain and fusing a T4-foldon sequence at the C-terminus. The gene sequence encoding the recombinant hemagglutinin protein is shown in SEQ ID NO.

2.

4. The use of the recombinant hemagglutinin protein as described in any one of claims 1-3 in the preparation of subunit vaccines.

5. A subunit vaccine, characterized in that, The recombinant hemagglutinin protein described in any one of claims 1-3 is used as the antigen.

6. A method for preparing the recombinant hemagglutinin protein according to any one of claims 1-3, characterized in that, The nucleotide sequence encoding recombinant hemagglutinin protein was cloned into a vector to obtain a recombinant plasmid, which was then transfected into cells and expressed and purified to obtain recombinant hemagglutinin protein.

7. The method for expressing recombinant hemagglutinin protein according to claim 6, characterized in that, The carrier is a pEE12.4 carrier; The recombinant plasmid was transfected into CHO cells and expressed, then purified to obtain recombinant hemagglutinin protein.

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