CN117683098A

CN117683098A - HA truncated protein of recombinant avian influenza virus H9N2 subtype, preparation method and application thereof

Info

Publication number: CN117683098A
Application number: CN202211100214.7A
Authority: CN
Inventors: 吴素芳; 张强; 车影; 钱泓; 吴有强; 闻雪; 贾宝琴; 李冰飞
Original assignee: Novo Biotech Corp
Current assignee: Novo Biotech Corp
Priority date: 2022-09-09
Filing date: 2022-09-09
Publication date: 2024-03-12

Abstract

The invention discloses a recombinant avian influenza virus H9N2 subtype HA truncated protein, a preparation method and application thereof. The avian influenza virus H9N2 subtype HA truncated protein utilizes a genetic engineering technology to construct a pEE12.4-AIV-HA (H9) vector, stably transfects and screens to obtain a CHO cell strain which efficiently secretes and expresses HA antigen protein, and the subunit vaccine is prepared by emulsifying the purified HA protein mixed adjuvant. The subunit vaccine prepared by the HA truncated protein of the avian influenza virus H9N2 subtype HAs high antigen stability, high purity and strong specificity, and provides a new thought for the research and development of the avian influenza virus H9N2 subunit vaccine.

Description

HA truncated protein of recombinant avian influenza virus H9N2 subtype, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological products, and relates to an HA truncated protein of a recombinant avian influenza virus H9N2 subtype, a preparation method thereof and application of the HA truncated protein in preparation of vaccines.

Background

Avian Influenza (AI) is a highly contagious disease caused by influenza a virus (AIV), AIV is classified into 18 subtypes (H1-H18) according to the antigenic difference of Hemagglutinin (HA), 11 subtypes (N1-N11) according to the difference of Neuraminidase (NA), and influenza viruses of different subtypes can be classified into highly pathogenic virus (HPAIV) and less pathogenic virus (LPAIV) according to their intensity of pathogenicity. H9N2 belongs to LPAIV, and can cause respiratory symptoms, laying rate, fertilization rate reduction and the like of poultry and secondary infection, and the proportion of H9N2 avian influenza subtype infected by chicken flock in 1996-2000 is 93.89%, although with large-scale inoculation of vaccine and upgrading of epidemic prevention policy, investigation in south China shows that positive samples of H9N2 influenza viruses still occupy 78.18% of positive samples in 2013-2018, so H9N2 is one of main avian influenza virus subtypes except H5N1 and H7N 9.

The large-scale infection of H9N2 not only provides a wide gene library for other influenza viruses, but also has research on the part of H9N2 internal genes can enhance the pathogenicity of other viruses. For example, substitution of the PA and NP genes may enhance the pathogenicity of H5N 1; the internal genes of the recombinant H7N9 avian influenza virus exploded in 2013 are evidence to come directly from H9N2; H1N1 and H3N2 can also be widely recombined with H9N 2. In addition, H9N2 gradually acquires the capability of directly infecting a human after extensive antigen drift and antigen conversion, so the method is important for monitoring and preventing H9N 2.

Like other viral diseases, vaccination is still the currently most effective AIV control measure. The first H9N2 inactivated vaccine is approved for use since 1998, and after that, the monovalent vaccine and the multi-linked vaccine are marketed sequentially, thereby playing an important role in preventing large-scale AIV diffusion in China. The inactivated vaccine has higher safety and convenient preservation, is the most common vaccine form, but antigen protein can generate loss in the inactivation process, so that the normal inoculation amount is larger, and an adjuvant is added to achieve the immune effect, so that the cost of the inactivated vaccine is increased, certain side effects are brought, and the development of an efficient, safe and practical avian influenza vaccine is urgent.

Since the advent of avian influenza virus, it has been a great hazard to human production and life. H9N2 has also received extensive attention as one of the most dominant subtypes, and in order to prevent H9N2 pandemic, humans have developed various monovalent or multiple vaccines, and the production process of the first generation vaccines produced in attenuated or inactivated form is simple, but the production period is long, and the production period depends on chick embryo and interferes with serological detection and epidemiological investigation. Aiming at the defect, most of novel second-generation vaccines utilize genetic engineering means to produce recombinant vector vaccines or subunit vaccines, solve the problems of chicken embryo dependence, interference of serological detection, epidemiological investigation and the like, and the common recombinant vector vaccine production system expresses influenza virus protective antigen by means of chicken pox virus, newcastle disease virus and lactobacillus plantarum, and can induce protective immunity after entering a host body. The production principle of subunit vaccine is that protective antigen HA of influenza virus is directly produced by means of prokaryotic or eukaryotic expression system, hemagglutinin (HA) is used as main surface antigen of influenza virus, and is responsible for combination and entering host cell, can induce and produce neutralizing antibody, and can stimulate organism to produce cellular immunity.

Although development of subunit vaccines against HA proteins HAs been a focus of research on influenza virus vaccines, there are limitations of the various expression systems currently in use. The escherichia coli expression system HAs high efficiency of producing the HA protein, but the produced HA protein HAs poor immunogenicity due to the lack of glycosylation modification capability, and the HA protein is easy to appear in the form of inclusion bodies, so that the HA protein is not beneficial to the purification and large-scale production of the protein. Baculovirus-insect cell expression systems can produce glycosylated HA proteins, but the protein expression levels are low, making it difficult to form seed banks that stably express HA proteins.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an HA truncated protein of a recombinant avian influenza virus H9N2 subtype, which is: (1) the amino acid sequence is SEQ ID NO.2; or (2) a protein which is formed by deleting, replacing or adding one or more amino acids on the protein with the amino acid sequence of SEQ ID NO.2 and has the function of the protein in the protein with the amino acid sequence of SEQ ID NO. 2.

In a preferred embodiment of the HA truncated protein of the present invention, the HA truncated protein preferably comprises a tag. More preferably, the tag is a 6×His tag, and the amino acid sequence of the tag is shown in SEQ ID NO. 3.

In a preferred embodiment of the HA truncated protein of the present invention, preferably, the nucleotide sequence of the HA truncated protein is shown in SEQ ID No. 1.

According to another aspect of the present invention there is provided a vaccine composition comprising the HA truncated protein of the present invention and a pharmaceutically acceptable adjuvant.

According to a further aspect of the present invention there is provided a method of preparing a truncated HA protein according to the present invention, the method comprising the steps of:

1) Cloning the HA truncated protein encoding gene of the avian influenza virus H9N2 subtype subjected to codon optimization into a eukaryotic expression vector as shown in SEQ ID NO.1 to obtain a recombinant plasmid containing the HA truncated protein encoding gene of the avian influenza virus H9N2 subtype;

2) Then, the recombinant plasmid containing the HA truncated protein coding gene of the H9N2 subtype avian influenza virus is transfected into CHO cells to obtain CHO cell strains;

3) Obtaining a highly expressed cell strain by culturing, screening and domesticating the CHO cell strain in the step 2); and

4) And (3) fermenting and culturing the cell strain with high expression in the step (3), and purifying to obtain the HA truncated protein of the H9N2 subtype of the recombinant avian influenza virus.

In a preferred embodiment of the preparation method of the present invention, the eukaryotic expression vector is preferably pee6.4, pee12.4, pgl4.13, pcdna3.1. More preferably, the eukaryotic expression vector is pee12.4.

In a preferred embodiment of the production method of the present invention, the CHO cell line is preferably CHO-K1.

According to a further aspect of the invention, the invention provides the use of a truncated protein of HA of the H9N2 subtype of avian influenza virus for the preparation of a vaccine or diagnostic reagent.

By analyzing the influenza virus HA protein sequence (GenBank: MF 794999), it was found that the genomic sequence 28-1707 was an avian influenza virus HA protein sequence. Further analysis 1M-18A may be the secretion signal peptide of the HA protein, 19D-524N may be the extracellular domain of the HA protein, 525L-547W may be the transmembrane domain of the HA protein, 548A-560I may be the intracellular domain protein of the HA protein. By combining the experience of our earlier expression virus envelope protein research, the C-terminal sequence which can affect the stability of the recombinant protein is further removed from the extracellular region 507E-524I through the protein structure prediction and the research of the structure of the amino acid itself. We select CHO cell to express the amino acid sequences of extracellular region 1M-506L and extracellular region 1M-524I of HA protein, and find that extracellular region 1M-506L can obviously increase the secretion expression quantity of HA protein, and the amino acid sequence of the mature HA protein of 19D-506L is listed in the sequence table because the mature HA protein does not include a signal peptide part.

The invention utilizes genetic engineering technology to stably transfect HA protein gene into CHO cells, obtains CHO cell strain capable of stably and highly expressing HA protein through screening, and develops subunit vaccine aiming at H9N2 avian influenza virus by mixing and emulsifying HA protein with an adjuvant after purification. Animal experiments prove that the inoculated amount of the HA protein reaches 50 mug each, and the SPF chickens which are subjected to H9N2 challenge can be completely protected. The HA protein is produced by using mammalian cell CHO as expression vector, which not only solves the problem of protein glycosylation modification. Furthermore, a seed pool stably expressing the HA protein can be preserved. In addition, CHO cells seldom secrete endogenous proteins and have the extracellular secretion function of products, which is beneficial to the purification of proteins and the mass production of vaccines.

In conclusion, the subunit vaccine prepared from the HA truncated protein of the avian influenza virus H9N2 subtype HAs high antigen stability, high purity and strong specificity, lays a foundation for the development of the technology for expressing the foreign protein by the CHO system, and provides a new thought for the research and development of the subunit vaccine of the avian influenza virus H9N 2.

Drawings

FIG. 1 shows the electrophoresis pattern of HA gene PCR amplification products, M: DL5000marker,1, 2: a fragment of interest AIV-HA (H9);

FIG. 2 shows an electrophoresis chart for PCR verification of recombinant plasmid bacterial liquid, M: DL5000marker,1-6: pEE12.4-AIV-HA (H9) clone No. 1-6, target fragment about 1788bp, positive clone, 7: pee12.4-AIV-HA (H9) negative control;

FIG. 3 shows an electrophoretogram for recombinant plasmid restriction enzyme assay, M: DL10000marker,1-3: the pEE12.4-AIV-HA (H9) plasmid No. 1-3 HAs double enzyme cutting bands, the sizes of which are 8746bp and 1551bp respectively;

FIG. 4Western blot analysis of HA protein expression, M: protein relative molecular mass standard, 1: plasmid pEE12.4-AIV-HA (H9) (1M-506L) 96H transfection supernatant, 2: plasmid pEE12.4-AIV-HA (H9) (1M-524I) 96H transfection supernatant

FIG. 5 shows SDS-PAGE analysis of HA protein expression, M: protein relative molecular mass standard, 1, monoclonal No. 1; 2, monoclonal No. two; 3, monoclonal No. three; 4, monoclonal No. four; 5, fifth monoclonal;

FIG. 6 shows the serum HI antibody titers of SPF chickens after immunization;

FIG. 7 shows the detoxification of SPF chickens 5 days after detoxification.

Detailed Description

1. Materials and methods

1.1 materials

1.1.1 viruses, vectors and cells

Avian influenza virus subtype H9 (A/Chicken// Hebei/H9N 2/2015) Chicken embryoid bodies, pUC57 vector, pEE12.4 vector and CHO-K1 cells were supplied by Zhejiang Hailong Biotechnology Co.

1.1.2 chick embryo and laboratory animal

10 day old SPF chicks 400-800, 4-5 week old SPF chicks 35 were purchased from Experimental animal technologies Inc. of Beijing Bolin and Yingrah.

1.1.3 major reagents

HindIII and EcoRI restriction enzymes were purchased from NEB, inc. of the United states;

t4 DNA ligase and gel recovery kit were purchased from TAKARA company;

plasmid miniprep kit was purchased from OMEGA company;

the endotoxin-free large-scale kit is purchased from QIAGEN company;

fetal bovine serum was purchased from Hyclone company;

pancreatin 0.25% trypsin-EDTA, DMEM available from Gibco company;

Lipofectamine ^TM LTX, BCA protein quantification kit was purchased from Thermo Fisher, inc., USA;

sodium dihydrogen phosphate, sodium chloride, imidazole, tween-20, PMSF, benzamidine hydrochloride were purchased from biological engineering (Shanghai) Inc.;

the avian influenza virus H9 subtype HI test antigen and negative and positive serum are provided by animal epidemic disease research laboratories of animal husbandry and veterinary research institute of the national academy of sciences of Beijing and city.

1.1.4 primer design and Synthesis

Referring to the main H9N2 strain A/chicken/Daye/DY0602/2017GenBank popular in China in recent years: MF 794999), carrying out codon optimized synthesis by taking the HA sequence as a template, delivering the HA sequence to the nanjing golden sry company for synthesis, obtaining an AIV-SPV19-HA (H9) gene sequence (i.e., SEQ ID No. 1), and subcloning the synthesized sequence onto a pUC57 vector. Based on the nucleotide sequence, the up-and-down primers of HA gene (1M-506L) and (1M-524I) and the bacterial liquid PCR up-and-down primers were designed by SnapGene, and the sequences are as follows:

amplification of HA (1M-506L) and (1M-524I) upstream primers:

5＇-ACGAAGCTTGCCGCCACCATGATGAGGCCAATCGTGCTG-3＇

amplification of HA (1M-506L) downstream primer: 5' -TCGAATTCTCAATGGTGATGGTGATGGTGCAGCTTAGACTCCTCCT-3

Amplification of HA (1M-524I) downstream primer: 5' -CAATGAATTCTCAATGGTGATGGTGAT-3

Colony PCR upstream primers: 5' -GGAAGACTTAAGGCAGCGGC-3

Colony PCR downstream primers: 5 '-CAATGAATTCTCAATGGTGATGGTG-3'.

1.2 construction of recombinant plasmids

1.2.1 acquisition of the Gene of interest AIV-HA (H9) and the backbone vector pEE12.4

The target gene containing HindIII and EcoRI enzyme cutting sites is obtained by taking a synthetic vector pUC57-AIV-SPV19-HA (H9) as a template and using a designed primer for PCR amplification, after the correctness of the nucleic acid electrophoresis verification, the target gene is recovered by using the glue, and the product recovered by the glue and a skeleton vector are respectively subjected to double enzyme cutting, wherein the enzyme cutting system is as follows: 3-5. Mu.g of the gel was recovered with a carrier backbone, 5. Mu.L of each of 10x CutSmart buffer,Hind III-HF and EcoRI-HF, 2.5. Mu.L, and ddH was used ₂ 0 was added to 50. Mu.L and digested at 37℃for two hours. After the reaction, the gel was recovered by detecting the correct fragment by 1% agarose gel electrophoresis.

1.2.2 ligation and transformation

The gene of interest AIV-HA (H9) (1M-506L) was ligated to vector backbone pEE12.4 using T4 DNA ligase, the ligation system was as follows: 1. Mu.L of each of the backbone vector and the target gene, 1. Mu.L of T4 DNA ligase, 1. Mu.L of 10 XT 4 DNA ligase Buffer, and 1. Mu.L of the target gene were purified by ddH ₂ O was supplemented to 10. Mu.L and connected at 16℃for three hours. The ligation products were transformed and plated on ampicillin-resistant LB solid plates and incubated for 16 hours at 37℃in an inverted position.

1.2.3 screening of Positive clones

Dipping bacterial plaques growing on an Amp-resistant LB solid culture plate, and adding the following bacterial liquid PCR identification system: 7.5. Mu.L of 2 XTaq Master Mix, 0.3. Mu.L of bacterial liquid PCR upstream primer (10. Mu.M), ddH2O were supplemented to 15. Mu.L, the program was designed, the fragment of interest was amplified, and the correct positive clone was verified by nucleic acid electrophoresis and inoculated into 5mL LB medium containing Amp at 37℃at 220rpm/min and shaken overnight. The recombinant plasmid was extracted, digested and verified, and the error-free plasmid was further sequenced and verified and named pEE12.4-AIV-HA (H9) (1M-506L).

pEE12.4-AIV-HA (H9) (1M-524I) was constructed as 1.2.

1.3 extraction and transfection of plasmids

Recombinant plasmids pEE12.4-AIV-HA (H9) (1M-506L) and pEE12.4-AIV-HA (H9) (1M-524I) were extracted without endotoxin, and the concentration was measured and transfected. CHO-K1 cells were plated at 5X 10 at 24 hours prior to transfection ⁵ Spreading each well in 6-well plate, discarding culture medium when cell confluence reaches 60% -80%, washing with PBS for 2 times, adding 800 μl OPTI-MEM, and performing differential phase chromatographyLTX transfection reagent instructions respectively with 125 u L OPTI-MEM dilution of 2.5 u g recombinant plasmid and 7.5 u L transfection reagent LTX, then adding 2.5 u L plus, slowly blowing mixing after standing for 5 minutes, adding the recombinant plasmid into transfection reagent, slowly blowing mixing, standing for 10 minutes after slowly dropping into CHO-K1 cells, gently shaking the culture plate. DMEM medium changed to 10% fbs after 6 hours was cultured normally. The protein expression level was detected by western-blot at 96 hours after transfection.

1.4 pressure screening of monoclonal

The CHO-K1 cells were plated in 6-well plates and pressurized with different concentrations of puromycin, and the lowest puromycin concentration at which the cells all died after 5 days was the concentration of the pressurized screen. 48 hours after transfection, cell culture medium (dmem+10% fbs+1% diabody+puromycin) was changed, cells were all killed in the negative wells, the supernatant was collected, cells were digested and diluted to 1/well, 96 well plates were added, and after single cells were confluent in 96 well plates, the supernatant was collected.

1.5 identification of the expression level of the supernatant of the monoclonal cell line

Hemagglutination to detect HA protein expression, selecting higher hemagglutination titer fractions, further verifying by SDS-PAGE and Western Blot, performing amplification culture on cells of the high expression fraction, and collecting supernatant.

1.6 protein purification

Since the gene of interest is inserted into His tag, the protein can be purified by nickel column as follows: (1) sample preparation: the centrifuged cell supernatant was added with two protease inhibitors (PMSF mother liquor (1:1000) and benzamidine hydrochloride mother liquor (1:100)), tween-20 was added at a final concentration of 0.05% (v: v), and the mixture was uniformly mixed and filtered through a 0.8 μm filter membrane to obtain a sample. (2) pretreatment: all pipeline fillers are subjected to endotoxin removal treatment and soaked in 0.5M NaOH for at least 2 hours. (3) column equilibration: the filler is washed by ultrapure water until the pH value is neutral, and is balanced by Buffer A for 2-3 CV until the ultraviolet detection baseline is stable. (4) sample loading: and loading the sample, and collecting the flow-through liquid. (5) flushing: the column was washed with Buffer A and the UV detection baseline was stationary. (6) washing impurities: eluting the mixed protein by using Buffer B, collecting effluent liquid, and stabilizing to ultraviolet detection baseline. (7) elution: eluting protein with Buffer C, collecting effluent, and stabilizing to ultraviolet detection baseline. The fractions were subjected to SDS-PAGE.

The eluate containing the target protein was dialyzed against PBS, and the protein concentration was measured by the BCA method.

1.7 vaccine preparation

H9N2-HA protein expressed by CHO-K1 cells is purified, added with an adjuvant, emulsified, prepared into subunit vaccine with 200 mug/mL protein concentration, and used for animal experiments after sterile inspection.

1.8 animal test

1.8.1 immunization

35 SPF chickens of 4-5 weeks of age were selected and divided into 5 groups. Wherein, the immunization group 4 groups, 7 chickens in each group, respectively correspond to 100 mug/chicken, 50 mug/chicken, 25 mug/chicken and 6 mug/chicken, and the control group 1 group is set, and 7 chickens are injected with PBS as negative control. Immunization via chest intramuscular injection, first avoiding left chest, after 14 days of first avoiding, secondary immunization according to the same dosage and route, second avoiding right chest, blood sampling after 14 and 21 days of secondary immunization, and serum separation. HI antibodies were determined according to the method of chinese veterinary pharmacopoeia (2015 edition).

1.8.2 combating toxicity

Toxin was challenged 21 days after the second immunization, the toxin seeds were diluted 1:10 times with PBS (0.01M pH 7.4) and 0.2 ml/dose (about 2.5X10) by intravenous injection ⁷ EID 50). On day 5 after the challenge, each chicken throat and cloaca cotton swab is collected and placed in TPB (if the chicken throat and cloaca cotton swabs are stored at-80 ℃ for a long time) (if no toxicity is separated, the chicken throat and cloaca cotton swabs should be frozen at-20 ℃ immediately, and the influence of freezing and thawing on influenza viruses is not great). Taking each SPF chicken throat and cloaca cotton swab TPB (thawing in advance, enabling double antibodies to act at 4 ℃), inoculating 5 SPF chicken embryos of 10 days old into each allantoic cavity, 0.2 mL/embryo, incubating at 37 ℃ for 96 hours (non-specific death is achieved within 24 hours, and the dead chicken embryos are preserved at 4 ℃ for at most one day for measuring the antibodies), measuring the HA titer of allantoic fluid of all chicken embryos, and judging that virus separation is positive if the HA titer of allantoic fluid of 1 chicken embryo is not less than 1:16 in the chicken embryos inoculated by each swab sample. For virus isolation negative samples, the HA titer of chick embryo allantoic fluid should be determined after 1 generation of blind transmission.

2. Results

2.1 cloning of the fragment of interest

The pUC57-AIV-HA (H9) is used as a template, a target gene (1M-506L) with HindIII and EcoRI enzyme cutting sites is amplified by PCR, the detection of 1% agarose gel electrophoresis is shown in figure 1, figure 1 is an electrophoresis diagram of PCR amplification products of HA genes, and the method comprises the following steps of: AIV-HA (H9) target fragment. As can be seen from fig. 1: the band size was about 1565bp, which was expected.

2.2 bacterial liquid PCR verification

Picking single colony of pEE12.4-AIV-HA (H9) (1M-506L) obtained by connection transformation, performing colony PCR amplification, spotting amplified fragments, performing 1% agarose gel electrophoresis detection, and obtaining a result shown in FIG. 2, wherein FIG. 2 is a PCR verification electrophoresis diagram of recombinant plasmid bacterial liquid, and M is DL5000marker,1-6: pEE12.4-AIV-HA (H9) (1M-506L) clone No. 1-6, the target fragment about 1788bp, both positive clones, 7: negative control. As can be seen from FIG. 2, the band size is about 1788bp, which is expected.

2.3 double enzyme digestion identification

Randomly picking 3 PCR positive clones pEE12.4-AIV-HA (H9) (1M-506L), double digestion of miniplasmids with Hind III and EcoR I, electrophoresis, and showing two bands, the results of pEE12.4 vector (8746 bp) and AIV-HA (H9) (1M-506L) target gene (1551 bp) are shown in FIG. 3, FIG. 3 is a recombinant plasmid enzyme digestion verification electrophoresis diagram, M: DL10000marker,1-3: pEE12.4-AIV-HA (H9) (1M-506L) plasmid No. 1-3 double digested strips, the sizes of which are 8746bp and 1551bp respectively. As can be seen from fig. 3, it is expected. The sequencing result shows that the AIV-HA (H9) (1M-506L) gene sequence is correct.

2.4 CHO-K1 cell transient detection

After the plasmid is transfected into CHO-K1 cells, the expression of HA protein in the transfected supernatant is detected by Western blot for 96 hours, the result is shown in FIG. 4, the expression of HA protein is analyzed by Western blot in FIG. 4, and M: protein relative molecular mass standard, 1: plasmid pEE12.4-AIV-HA (H9) (1M-506L) 96H transfection supernatant, 2: plasmid pEE12.4-AIV-HA (H9) (1M-524I) 96H transfected supernatant. As can be seen from FIG. 4, the expression of HA protein was detected in 96h supernatant, about 85kDa, 55.6kDa higher than the theoretical predicted value, but the actual size of the glycosylated protein was met, and the protein expression level of the expressed HA (1M-506L) protein was significantly higher than that of HA (1M-524I).

2.5 detection of expression of HA protein by monoclonal cell lines

After the monoclonal cells in the 96-well plate are cultured to 80% -90% of confluency, the supernatant of each well is collected for SDS-PAGE detection, the result is shown in FIG. 5, and the result is shown in FIG. 5, wherein the SDS-PAGE analysis shows HA protein expression, M: protein relative molecular mass standard, 1, monoclonal No. 1; 2, monoclonal No. two; 3, monoclonal No. three; 4, monoclonal No. four; 5 No. five monoclonal. As can be seen from FIG. 5, the No. 1-5 monoclonal cell lines all have HA protein expression, and the No. 5 strain HAs the highest expression level.

2.7 antibody level detection

HI antibody titers of 14d and 21d after secondary immunization are shown in fig. 6, and fig. 6 is the post-immunization SPF chicken serum HI antibody titers. As can be seen from FIG. 6, the lowest dose immunization (12. Mu.g/mL) induced detectable HI antibody in addition to the control, and the different immunization dose groups produced different HI antibody titers and were dose dependent. The average HI antibody titer can reach 2.71log2 at 14d after the second immunization, and reaches 5.71log2 at 21d after the second immunization.

2.8 toxin expelling detection

5d after the virus is attacked, collecting a throat swab, detecting the virus expelling condition of a test animal, inoculating 9-10 days old SPF chick embryo after the swab is placed in a sterile TPB, detecting the allantoic fluid hemagglutination condition of the chick embryo after 96h, judging the virus expelling condition of the SPF chick, and as shown in figure 7, wherein figure 7 is the virus expelling condition of the SPF chick after 5 days of virus attack. As can be seen from FIG. 7, the immunization dose reached 50. Mu.g and above per component, induced complete immune protection, and achieved complete inhibition of viral replication 5d after viral challenge.

The present invention is illustrated by the examples above, but it should be understood that the invention is not limited to the specific examples and embodiments described herein. These specific examples and embodiments are included herein for the purpose of aiding those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention, and therefore the invention is limited only by the content and scope of the appended claims, which are intended to cover all alternatives and equivalents that are included within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A recombinant avian influenza virus H9N2 subtype HA truncated protein, characterized in that the HA truncated protein is:

(1) The amino acid sequence is SEQ ID NO.2; or (b)

(2) And deleting, replacing or adding one or more amino acids on the protein with the amino acid sequence of SEQ ID NO.2 to form a protein with the protein function in the protein with the amino acid sequence of SEQ ID NO. 2.

2. The HA-truncated protein of claim 1, wherein the HA-truncated protein comprises a tag.

3. The HA-truncated protein of claim 2, wherein the tag is a 6 x his tag having the amino acid sequence shown in SEQ ID No. 3.

4. The HA-truncated protein of claim 3, wherein the nucleotide sequence of the HA-truncated protein is shown in SEQ ID No. 1.

5. A vaccine composition comprising the HA truncated protein of any one of claims 1-4 and a pharmaceutically acceptable adjuvant.

6. A method for preparing a HA truncated protein according to any one of claims 1 to 4, comprising the steps of:

2) Then, the recombinant plasmid containing the HA truncated protein coding gene of the avian influenza virus H9N2 subtype is transfected into CHO cells to obtain CHO cell strains;

7. The method of claim 6, wherein the eukaryotic expression vector is pEE6.4, pEE12.4, pGL4.13, pcDNA3.1.

8. The method of claim 7, wherein the eukaryotic expression vector is pee12.4.

9. The method according to claim 6, wherein the CHO cell line is CHO-K1.

10. Use of a HA truncated protein of the H9N2 subtype of avian influenza virus according to any one of claims 1 to 4 for the preparation of a vaccine or diagnostic reagent.