CN113913395A

CN113913395A - Artificial recombinant H5N8 influenza virus and preparation method and application thereof

Info

Publication number: CN113913395A
Application number: CN202111217660.1A
Authority: CN
Inventors: 陈化兰; 曾显营; 李雁冰; 邓国华; 田国彬; 李呈军
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2022-01-11
Anticipated expiration: 2041-10-19
Also published as: CN113913395B

Abstract

The invention discloses an artificial recombinant H5N8 influenza virus, a preparation method and application thereof. The artificially recombined H5N8 influenza virus HAs high pathogenicity avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) surface antigen Hemagglutinin (HA) and Neuraminidase (NA) genes, but HA cleavage sites present molecular characteristics (-RETR-) of typical low pathogenicity avian influenza virus, HAs 6 internal genes of PB2, PB1, PA, NP, M and NS of influenza virus chick embryo high titer adaptive strain A/PR/8/34(H1N1), HAs the strain number of H5-Re14, and HAs the preservation number of CCTCC NO: v202160. The chicken embryo adaptability is good, and the virus growth titer can be improved by more than 9 times compared with that of wild virus; the vaccine strain is used for producing the vaccine, so that the production cost of the vaccine can be greatly reduced, and the quality of the vaccine is improved.

Description

Artificial recombinant H5N8 influenza virus and preparation method and application thereof

Technical Field

The invention relates to the technical field of reverse genetics and animal infectious diseases, in particular to an artificially recombined and weakened H5N8 subtype recombinant influenza virus and a preparation method and application thereof.

Background

Avian Influenza Virus (AIV) is a segmented negative-strand RNA virus that can infect a wide range of birds and mammals. AIV is classified into 16 Hemagglutinin (HA) and 9 Neuraminidase (NA) subtypes based on the surface proteins of the virus. Among them, AIV subtype H5 and H7 may cause epidemic in poultry or wild birds, resulting in significant economic loss, and may also infect humans, causing public health hazards. China developed a series of full virus inactivated vaccines, successfully controlled H5N1 since 2004 and H7N9 subtype avian influenza since 2017.

In order to deal with the poultry H5N8 epidemic situation and public health hazard which may appear in China, the invention develops the artificial recombinant H5N8 avian influenza vaccine strain.

The ideal influenza virus vaccine strain should have good antigen matching with epidemic strains, no pathogenicity to chicken embryo, high-titer growth of chicken embryo and other conditions. However, it is difficult to isolate influenza strains having the above conditions from nature as vaccine strains.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides an artificial recombinant H5N8 influenza virus, a preparation method and application thereof.

The technical scheme of the invention is as follows: the recombinant H5N8 avian influenza virus takes highly pathogenic H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) as surface antigen Hemagglutinin (HA) and Neuraminidase (NA) gene donors, but the HA cleavage site HAs the molecular characteristics (-RETR-) of the typical low pathogenic avian influenza virus; the PB2, PB1, PA, NP, M and NS genes of an influenza virus chick embryo high-titer adaptive strain A/PR/8/34(H1N1) are donors of 6 internal genes, and the H5N8 avian influenza virus of 1 strain recombination is obtained by artificial recombination through a reverse genetic operation method, is named as influenza A virus A/Harbin/H5-Re14/2021(H5N8) (abbreviated as H5-Re14 strain), is preserved in China center for type culture Collection, is addressed to Wuhan university in Wuhan, China, and has the preservation number of CCTCC NO: v202160, preservation time 2021, 8 months and 3 days.

The invention selects 6 internal genes of laboratory chick embryo adaptive strain A/PR/8/34(H1N1) of influenza virus which can grow to high titer in chick embryo as skeleton genes, selects HA and NA genes of highly pathogenic avian influenza virus strain A/swan/Shanxi/4-1/2020(H5N8) separated in 2020 as surface genes, and utilizes a reverse genetics artificial construction method to prepare a attenuated recombinant virus H5-Re14 strain which can be used as a vaccine strain of H5N8 influenza.

Experiments prove that the H5-Re14 strain virus has consistent antigenicity with the avian influenza virus epidemic strain A/swan/Shanxi/4-1/2020(H5N8) of H5N8 in China, and after the vaccine is prepared for immunizing chickens, a good specific protection effect can be generated on the attack of the avian influenza virus epidemic strain A/swan/Shanxi/4-1/2020(H5N8) of H5N 8. The virus contains 6 internal genes of an influenza virus chick embryo high-titer adaptive strain A/PR/8/34(H1N1), so that the virus has good chick embryo adaptability, and the virus growth titer can be improved by over 9 times compared with wild virus; the vaccine strain is used for producing the vaccine, so that the production cost of the vaccine can be greatly reduced, and the quality of the vaccine is improved.

Furthermore, the invention also provides application of the recombinant H5N8 avian influenza virus (H5-Re14 strain) in preparation of a medicament for preventing diseases caused by the H5N8 influenza virus.

Further, the H5N8 influenza virus is A/swan/Shanxi/4-1/2020(H5N8) strain.

Furthermore, the invention also provides a method for constructing the recombinant H5N8 avian influenza virus, which comprises the following steps:

(1) HA1 and HA2 gene segments of highly pathogenic H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) are amplified by using HA gene specific amplification and mutation primers and an RT-PCR method, and HA1 segments are amplified while amino acid at the cracking site of HA gene is mutated from-RERKR-to-RETR-so that molecular characteristics of highly pathogenic avian influenza virus are mutated into molecular characteristics of typical low pathogenic avian influenza virus; then, an HA segment with a cracking site being the molecular characteristics of the typical low-pathogenicity influenza virus is amplified by utilizing an overlap extension reaction, and a pBD plasmid is inserted to construct a recombinant HA gene bidirectional transcription vector;

(2) amplifying NA gene full gene segments of H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) by using an RT-PCR method, inserting pBD plasmids, and constructing a recombinant NA gene bidirectional transcription vector;

(3) mixing 2 recombinant pBD bidirectional transcription vector plasmids of HA and NA genes constructed in the steps (1) and (2) with 6 pBD bidirectional transcription vector plasmids of PB2, PB1, PA, NP, M and NS genes of an influenza virus chick embryo high-titer adaptive strain A/PR/8/34(H1N1), inoculating 293T cells with a transfection reagent, inoculating chick embryos to cells harvested after culture and a supernatant to obtain the H5N8 avian influenza rH5N8-2/6 recombinant virus strain with HA activity. The obtained recombinant virus HA and NA are from a domestic isolate A/swan/Shanxi/4-1/2020(H5N8) of highly pathogenic H5N8 influenza virus, but HA cleavage sites present the molecular characteristics (-RETR-) of a typical low-pathogenicity avian influenza virus, and 6 internal genes PB2, PB1, PA, NP, M and NS are from an influenza virus chick embryo high-titer adaptive strain A/PR/8/34(H1N 1).

Further, a primer pair for amplifying the HA1 gene is shown as SEQ ID No.9 and SEQ ID No.10, wherein the SEQ ID No.10 is a mutation primer of an HA cleavage site; the primer pair for amplifying the HA2 gene is shown as SEQ ID No.11 and SEQ ID No. 12.

Compared with the prior art, the invention has the following beneficial effects:

1. H5-Re14 has the same antigenicity with the avian influenza virus epidemic strain A/swan/Shanxi/4-1/2020(H5N8) of H5N8 in China, and after the vaccine is prepared for immunizing chickens, a good specific protection effect is generated on the attack of the avian influenza virus epidemic strain A/swan/Shanxi/4-1/2020(H5N8) of H5N 8. The virus contains 6 internal genes of influenza virus chick embryo high titer adaptive strain A/PR/8/34(H1N1), so that the virus has good chick embryo adaptability, and the virus growth titer can be improved by over 9 times compared with wild virus; the vaccine strain is used for producing the vaccine, so that the production cost of the vaccine can be greatly reduced, and the quality of the vaccine is improved.

2. Through artificial modification of HA gene cleavage sites, the H5-Re14 strain virus HAs the molecular characteristics of typical low-pathogenicity influenza virus, the H5-Re14 strain recombinant virus HAs no pathogenicity to chicken embryos and chickens, and the biological safety is high.

Drawings

FIG. 1 is a gel electrophoresis diagram of RT-PCR amplified genes, wherein A is HA1, HA2, HA and NA gene fragments amplified by RT-PCR with A/swan/Shanxi/4-1/2020(H5N8) virus strain cDNA as a template, which are respectively lanes 1-4, and M is DNA Marker 2000; b is 8 gene segments of the recombinant virus amplified by RT-PCR by taking cDNA of the recombinant H5N8 virus as a template, lanes 1 to 8 are HA, NA, PB2, PB1, PA, NP, M and NS gene segments respectively, and M is DNA Marker 2000.

FIG. 2 shows the sequences before and after mutation of HA gene cleavage site region of H5-Re14 virus.

FIG. 3 shows growth curves of recombinant H5-Re14 virus and wild type H5N8 influenza A/swan/Shanxi/4-1/2020(H5N 8).

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Experimental Material

1. Viral strains

A/swan/Shanxi/4-1/2020(H5N8) (SW/SX/4-1/20 for short) is separated from Shanxi dead swan in China in 2020, and is separated, identified and stored by the avian influenza reference laboratory in Harbin veterinary institute of Chinese academy of agricultural sciences, wherein the strain is recorded in the literature: "the immunity efficacy research of the prior recombinant trivalent inactivated vaccine against the H5 and H7 virus antigen variants" of the recombinant avian influenza virus (H5+ H7) "Liuyanjing et al, the Chinese veterinary medicine bulletin, 9 months of Jiejiejing in 2021, was published on line. The applicant has guaranteed that the biomaterial is openly available within twenty years from the filing date.

SPF chick embryos and cells

SPF-chicken embryos and SPF-chickens, purchased from Harbin veterinary institute of Chinese academy of agricultural sciences/national avian laboratory resources Bank, 293T cells (human embryonic kidney cells, SCSP-502) were purchased from the cell Bank of the culture Collection of representatives of Chinese academy of sciences.

3. Plasmid vector

pBD vectors (Zejun Li, Hualan Chen, Pearong Jiao, Guohua Deng, Guobin Tian, Yanbin Li, Erich Hoffmann, Robert G.Webster, Yuniko Matsuoka, and Kangzhen Yu.molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse model.J.Virol.2005, 79:12058-12064) were constructed by aged blue researchers, and XbaI cleavage fragments containing the polymerase I promoter-Sapi cloning site-murine ribozyme sequence were included in the vector for unidirectional transcription plasmid, XbaI cleavage fragments were inserted in the pCI (Promega) plasmid in reverse direction to form the RNA polymerase II promoter (CMV) transcription termination signal sequence Rib → RNA genome → mRNA (SV 5 → 3) for bidirectional transcription of the RNA polymerase (mRNA) and mRNA (mRNA) for bidirectional transcription of the vector for RNA polymerase RNA termination → RNA transcription could be constructed. pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, pBD-M, and pBD-NS vectors inserted into 6 internal genes such as A/PR/8/34(H1N1) strains PB2, PB1, PA, NP, M, and NS were identified and stored by avian influenza reference laboratory in Harbin veterinary institute of Chinese academy of agricultural sciences (Guobin Tian, Suhua Zhuang, Yanbig Li, Zhigao Bu, Peahong Liu, Jiyong Zhou, Chengjun Li, Jianzhong Shi, Kangzhen Yu, Hugan Chen.

4. Primary reagent

The virus RNA extraction kit is purchased from Beijing Tiangen Biochemical technology Co., Ltd; a reverse transcription Kit (ReverTra Ace RT Kit) was purchased from Toyobo, Japan; ex-taq enzyme was purchased from Takara; PCR product purification KIT (CYCLE-PURE-KIT) was purchased from Omega; gel recovery kit (Wizard SV Gel and PCR Clean-Up System) was purchased from Promega; sequencing kit (ABI BigDye Terminator v3.1) was purchased from Thermo Fisher, USA; plasmid extraction Kit (QIAGEN Plasmid Midi Kit) was purchased from QIAGEN.

5. Primer and method for producing the same

Analysis and comparison, specific primers of synthetic H5N8 influenza isolate SW/SX/4-1/20(H5N8) HA1, HA2 and NA gene were designed, and the sequences of the synthetic primers are shown in Table 1.

TABLE 1 HA and NA Gene specific primer sequences for H5N8 avian influenza Virus SW/SX/4-1/20(H5N8)

Analysis and comparison, designing specific primers of PB2, PB1, PA, NP, M and NS genes for synthesizing A/PR/8/34(H1N1), and synthesizing primer sequences shown in Table 2.

TABLE 2 primer sequences specific for 6 internal gene segments of influenza A/PR/8/34(H1N1)

Example 1: preparation and identification of recombinant H5N8 influenza virus H5-Re14

1. Construction and identification of 2 surface genes (HA and NA) of recombinant viruses:

extracting RNA of an H5N8 subtype highly pathogenic avian influenza isolate SW/SX/4-1/20(H5N8), and reverse-transcribing the cDNA into cDNA, using HA1 fragment-specific primers (HA 1-U: atg gag aac ata gta ctt ctt ctt g; HA 1-L-: gcc ccg aac agg cct cta gtt tct ctt aga gga cta ttt ctg agc, wherein HA1-L is a primer of which the mutant HA cleavage site is the molecular characteristic of low pathogenic virus) and HA2 fragment-specific primers (HA 2-U: act aga ggc ctg ttc ggg gcg ata gca ggg t; HA 2-L: tta aat gca aat tct gca ctg taa c) of the HA gene, respectively amplifying HA1 and HA2 fragments of the HA gene (A in figure 1) by using an RT-PCR method, carrying out weakening mutation modification on an HA cleavage site while amplifying an HA1 gene segment; then, HA1 and HA2 are used as templates, HA1-U/HA2-L is used as a primer, phusion high fidelity polymerase is used for carrying out overlap extension reaction (SOE-PCR), and HA segments with cleavage sites showing the characteristics (-RETR-) of typical low pathogenicity avian influenza virus molecules are amplified (A in figure 1). And cloning the plasmid into pBD vector to constitute pBD-HA recombinant plasmid.

The mutant primer (HA1-L) of the HA gene cracking site is designed according to the specific sequence of a plurality of continuous basic amino acid sites of the HA gene, and the amplified HA gene cracking site is mutated from-REKRRKR-to-RETR-, so that the HA gene cracking site is mutated from the molecular characteristic of the highly pathogenic avian influenza virus to the molecular characteristic of the typical low pathogenic avian influenza virus (figure 2).

Then, the full length of the NA gene (A in figure 1) of the highly pathogenic H5N8 strain is amplified by an RT-PCR method and inserted into a pBD vector according to the method to construct a pBD-NA recombinant plasmid.

2. Rescue of recombinant virus:

the above-mentioned 2 recombinant plasmids for HA and NA genes and 6 recombinant plasmids for pBD-PB2, pBD-PB1, pBD-PA, pBD-NP and pBD-M, pBD-NS containing internal genes of A/PR/8/34(H1N1) virus were simultaneously introduced into 293T cells in a monolayer by lipofection, and after 48 hours, the cells and supernatant were harvested, and allantoic fluid was harvested after 48 hours and assayed for Hemagglutination (HA) activity by inoculating chick embryo allantoic cavities of 9 to 11 days old. The HA positive sample is the rescued H5N8 subtype low-pathogenicity recombinant virus, the obtained recombinant virus is named as A/Harbin/H5-Re14/2021(H5N8), the strain number is H5-Re14, the recombinant virus is preserved in the China center for type culture Collection of Wuhan university located in Wuhan, China, and the preservation number is CCTCC NO: v202160.

3. Sequence identification of recombinant viruses

Extracting RNA of the recombinant virus H5-Re14, respectively amplifying 8 segments of the recombinant virus H5-Re14 whole genome by using an RT-PCR method, carrying out nucleic acid gel electrophoresis (B in figure 1) on PCR products, and determining a specific sequence of each segment after gel recovery.

The HA gene sequence of the H5-Re14 strain is shown as SEQ ID No.1, the NA gene sequence of the H5-Re14 strain is shown as SEQ ID No.2, the PB2 gene sequence of the H5-Re14 strain is shown as SEQ ID No.3, the PB1 gene sequence of the H5-Re14 strain is shown as SEQ ID No.4, the PA gene sequence of the H5-Re14 strain is shown as SEQ ID No.5, the NP gene sequence of the H5-Re14 strain is shown as SEQ ID No.6, the M gene sequence of the H5-Re14 strain is shown as SEQ ID No.7, and the NS gene sequence of the H5-Re14 strain is shown as SEQ ID No. 8.

Comparing the sequence with the sequences of a wild strain H5N8 influenza virus domestic isolate SW/SX/4-1/20(H5N8) and influenza virus A/PR/8/34(H1N1) by using DNASAR software (DNASAR Lasergene V7.1), and finding that HA and NA of the recombinant viruses H5-Re14 come from the highly pathogenic H5N8 influenza virus domestic isolate SW/SX/4-1/20(H5N8), but the HA cleavage site HAs the molecular characteristics (-RETR-); the 6 internal genes PB2, PB1, PA, NP, M and NS are derived from the highly adapted influenza virus A/PR/8/34(H1N1) in chicken embryos.

4. Determination of growth curve of recombinant virus H5-Re14

Avian influenza vaccines are commonly produced from chicken embryos and require that the vaccine strain has good growth characteristics in the inoculated chicken embryos. The study was conducted to study the growth characteristics of the H5-Re14 strain virus in chicken embryos. Parent virus H5N8 influenza virus epidemic strain SW/SX/4-1/20(H5N8) and recombinant virus H5-Re14 are inoculated with 20 SPF chicken embryos of 2 groups respectively, the two groups of SPF chicken embryos are cultured for 24, 48, 72 and 96 hours respectively at the optimum 36 ℃, 5 chicken embryos cultured for different times are respectively taken for hemagglutination measurement, and the growth curves of the parent virus strain SW/SX/4-1/20(H5N8) and the recombinant virus H5-Re14 are detected.

As can be seen from the test results, the growth titer (average HA titer is 8.6log2 at most, namely 1:388) of the recombinant virus is obviously improved compared with that of the domestic isolate SW/SX/4-1/20(H5N8) (average HA titer is 5.4log2 at most, namely 1:42.2) of the parent H5N8 influenza virus under the culture condition of 36 ℃, and the hemagglutination titer is 9.19 times higher (figure 3). This shows that the recombinant virus with high growth titer prepared by the invention will bring great economic benefit for the subsequent vaccine batch production.

5. Antigenic analysis of recombinant virus H5-Re14

Antigenicity is a key index of whether viruses can be used as vaccine strains. In the research, a parent H5N8 influenza virus domestic isolate SW/SX/4-1/20(H5N8) and an artificially recombined H5N8 influenza virus are inactivated to prepare an oil emulsion inactivated vaccine, immune SPF chickens are prepared into antiserum, and an HI test is used for antigenicity analysis, wherein the HI test method refers to national standard (GB) of highly pathogenic avian influenza diagnosis technology (GB/T118936-2020)). The HI test result shows that the cross HI titer of the domestic isolate SW/SX/4-1/20(H5N8) of the parent H5N8 influenza virus and the artificially recombined H5N8 influenza virus and the corresponding serum is not different, which indicates that the virus still maintains the antigenicity of the parent strain after the H5N8 virus is recombined into the chick embryo adaptive H5-Re14 strain (see Table 3).

Table 3: antigenic analysis of parental strain SW/SX/4-1/20(H5N8) and recombinant virus H5-Re14 strain

6. Pathogenicity test of recombinant virus H5-Re14 strain on chick embryo and chick

The low pathogenicity of vaccine strains is a prerequisite for biosafety. SPF chick embryos of 9-11 days old and SFP chickens of 4-8 weeks old are often used as avian influenza virus pathogenicity evaluation models.

To understand the pathogenicity of recombinant strains, the invention first uses SPF chick embryos for assessment of viral pathogenicity. 20 SPF (specific pathogen free) chick embryos (purchased from Harbin veterinary institute of Chinese academy of agricultural sciences/national bird laboratory animal resource base) of 9 days old were randomly divided into 2 groups of 10, the first group was inoculated with 10 via allantoic cavity^-4The second group was inoculated 10 via allantoic cavity with double-diluted recombinant H5-Re14 strain virus fluid^-4Double dilution of the virus fluid of the parent strain SW/SX/4-1/20(H5N 8). Chick embryo death was observed and recorded within 72 hours after inoculation.

The invention simultaneously uses SPF chickens to evaluate the pathogenicity and the infection capacity of the virus. 20 SPF chickens of 6 weeks old are randomly divided into 2 groups, each group comprises 10 SPF chickens, the recombinant strain H5-Re14 and the parent strain SW/SX/4-1/20(H5N8) allantoic fluid are respectively diluted by 10 times, then the SPF chickens of 6 weeks old are inoculated by a vein way, the death condition of the SPF chickens is observed every day after inoculation, the disease index (IVPI) of the SPF chickens inoculated in the vein is calculated after 10 days of continuous observation. If the IVPI is more than 1.2, the strain is a highly pathogenic strain; if the IVPI is less than 1.2, the HA cleavage site is the molecular characteristic of the highly pathogenic avian influenza virus, and is a highly pathogenic avian influenza strain; if IVPI is less than 1.2, HA cleavage site is the molecular characteristic of low pathogenic influenza virus, then it is low pathogenic avian influenza virus strain.

20 SPF chickens of 6 weeks old are randomly divided into 2 groups of 10, the recombinant strain H5-Re14 and the parent strain SW/SX/4-1/20(H5N8) allantoic fluid are respectively diluted to 10⁶EID₅₀After each ml, 10 SPF-chickens 6 weeks old, 0.1 ml/chicken, were inoculated by the nasal route. And (3) observing the morbidity and mortality every day after inoculation, continuously observing for 14 days, simultaneously collecting laryngeal and cloaca swabs for virus titration, and evaluating the infection capacity of the virus to the chickens.

All test chickens were housed in negative pressure isolators with independent ventilation systems, and all tests involving the highly pathogenic H5N8 virus were conducted in a biosafety third-level laboratory.

The pathogenic test result of the SPF chick embryo shows that the chick embryo inoculated with the virus liquid of the parent strain SW/SX/4-1/20(H5N8) is completely killed within 26-30 hours after inoculation, and all the chick embryos inoculated with the virus liquid of the recombinant strain H5-Re14 are completely survived within 72 hours after inoculation, which shows that the recombinant virus of the H5-Re14 strain is not pathogenic to the chick embryo.

Test results of intravenous inoculation pathogenicity index (IVPI) determination of SPF chickens show that chickens inoculated with virus liquid of a parent strain SW/SX/4-1/20(H5N8) are all killed within 1 day after inoculation, and the IVPI of the chickens is 3.0; no adverse reaction occurs in all chickens inoculated with the recombinant strain H5-Re14 virus liquid within 10 days, and the constructed H5-Re14 recombinant virus IVPI is 0 (see Table 4). The H5-Re14 strain recombinant virus is shown to be a low-pathogenicity strain and has no pathogenicity to chickens.

The nasal infection test result of SPF chicken shows that all chicken inoculated with the virus liquid of the parent strain SW/SX/4-1/20(H5N8) through the nasal cavity die after being inoculated within 2-3 days; all chickens inoculated with the recombinant strain H5-Re14 virus solution through the nasal cavity have no adverse reaction within an observation period of 14 days; all chicken infected with SW/SX/4-1/20(H5N8) virus and dead chicken have positive larynx and cloaca swab virus detection, while chicken infected with H5-Re14 strain recombinant virus have negative virus detection in swab samples at 3 days and 5 days, which indicates that the H5-Re14 strain recombinant virus has no infection and pathogenicity to chicken (see Table 4).

The results show that the H5-Re14 strain recombinant virus has no pathogenicity to chicken embryos and chickens, and has high biological safety.

TABLE 4 pathogenicity and infectivity assay of recombinant viruses of strain H5-Re14

All the chickens in the group died after attacking the toxin within 2-3 days, and all the dead chickens detoxify and count the result of detoxifying 3 days after attacking the toxin; the group of chickens did not survive 5 days after challenge.

7. Immunogenicity assessment of recombinant Virus H5-Re14 Strain

A recombinant virus H5-Re14 strain with HA number of 8log2 is inactivated by 0.2% formaldehyde, and then an oil emulsion inactivated vaccine is prepared by taking American sornneborn brand-40 (Lytol) white oil as an adjuvant, 10 SPF (specific pathogen free) chickens with the age of 3 weeks are taken as model animals, 0.3 ml/muscle is injected for immunization, and 10 non-immune control chickens are arranged at the same time. HI antibody titers in all chicken sera were measured 21 days after immunization, simultaneously with 100CLD₅₀(CLD50 is half of death causing amount of chicken) attacks SW/SX/4-1/20(H5N8) for strong toxicity, observing 14 days after the toxicity is attacked, observing survival conditions, collecting swabs of larynx and cloaca at 3 days and 5 days after the toxicity is attacked, and inoculating chick embryos to detect toxicity expelling conditions. Dead chicken swab samples were collected at any time.

The results show that the HI antibody titer of the immune chicken serum against the H5N8 virus is between 7.0log2 and 9.0log2 at 21 days of immunization, the average titer is 8.0log2, and the control chicken serum is against the H5N8 diseaseThe antibody titers of the toxic HI are all less than 2.0log2 (HI is judged to be HI antibody negative by < 2log 2); nasal infection route 100LD 21 days after immunization₅₀After the dosage of the composition attacks the H5N8 subtype avian influenza virulent virus, all immunized chickens are healthy and alive, and virus detection of swab samples 3 days and 5 days after the attack is negative; the control chickens all died within 5 days after challenge, and virus isolation results of swabs of throat and cloaca of dead chickens are positive (see table 5).

TABLE 5 HI antibody after vaccine immunization of chicken prepared from recombinant virus strain H5-Re14 and survival and toxin expelling conditions after challenge

All the chickens in the group died after attacking the toxin within 2-4 days, all the dead chickens were detected to expel toxin, and the result of expelling the toxin was counted as the result of expelling the toxin 3 days after attacking the toxin; the group of chickens did not survive 5 days after challenge.

The results show that the artificial recombinant H5N8 influenza virus H5-Re14 has good immunogenicity and can prevent H5N8 subtype avian influenza.

Sequence listing

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)

<120> artificially recombined H5N8 influenza virus, preparation method and application thereof

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aatgttctac tgcatgcctt gagcatcata ttaatggtgt tagccctggg gaaaagtgaa 120

aacaatggaa tctgcaaggg aactatagta agggaatata atgaaacagt taggatagag 180

aaagtgaccc agtggtacaa cactagtgta gtcgaacatg taccgcattg gaacgagggc 240

gcttatataa acaacaccga accaatatgt gatgtcaagg gctttgcacc tttttccaag 300

gacaacggaa taagaattgg ctccagagga catatttttg tcataaggga gcctttcgtc 360

tcttgttcac ctgtagagtg cagaactttc ttcctcactc agggagctct actcaatgac 420

aaacactcaa atggaacagt gaaggatagg agcccattca gaactctcat gagtgtcgaa 480

gtgggtcaat cacccaatgt gtatcaagca aggtttgaag ctgtagcatg gtcagcaaca 540

gcctgtcatg atggtaagaa atggatgacg attggtgtga cagggccaga ttcgaaagca 600

atagcagtag tccattacgg aggagtgccc actgatattg ttaactcctg ggcaggagac 660

atattacgga ctcaggagtc atcttgtact tgcattcaag gtaattgtta ttgggtaatg 720

actgacggtc catccaatag acaggcgcag tatagaatat acaaagcaaa tcaaggcaaa 780

ataattgacc aagcagatgt cagctttagt ggagggcata ttgaggaatg ctcttgttat 840

ccaaatgatg gtaaagtgga atgcgtatgt agagacaact ggatgggaac taacaggcct 900

gtgctagtta tctcgcctga cctctcttac agggttgggt atttatgtgc gggattgccc 960

agtgacactc caagagggga agatgcccaa tttgtcggtt cgtgcactag tcccatggga 1020

aatcaggggt atggcgtaaa aggtttcggg tttcgacagg gaactgatgt gtggatgggg 1080

cggacaatta gtcgaacctc caggtcaggg tttgaaataa taaggataaa gaatggttgg 1140

acgcagacaa gcaaagaaca gattagaagg caggtggttg ttgataattt gaattggtcg 1200

ggatacagtg ggtctttcac tttaccagta gaattgtctg ggagggaatg tttagtcccc 1260

tgtttttggg tcgaaatgat cagaggcagg ccagaagaaa gaacaatctg gacctctagt 1320

agctccattg taatgtgtgg agttgatcat gaaattgccg attggtcatg gcacgatgga 1380

gctattcttc cctttgacat cgatgggatg taa 1413

<210> 3

<211> 2280

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaaagaa taaaagaact aagaaatcta atgtcgcagt ctcgcacccg cgagatactc 60

acaaaaacca ccgtggacca tatggccata atcaagaagt acacatcagg aagacaggag 120

aagaacccag cacttaggat gaaatggatg atggcaatga aatatccaat tacagcagac 180

aagaggataa cggaaatgat tcctgagaga aatgagcaag gacaaacttt atggagtaaa 240

atgaatgatg ccggatcaga ccgagtgatg gtatcacctc tggctgtgac atggtggaat 300

aggaatggac caatgacaaa tacagttcat tatccaaaaa tctacaaaac ttattttgaa 360

agagtcgaaa ggctaaagca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420

atacgtcgga gagttgacat aaatcctggt catgcagatc tcagtgccaa ggaggcacag 480

gatgtaatca tggaagttgt tttccctaac gaagtgggag ccaggatact aacatcggaa 540

tcgcaactaa cgataaccaa agagaagaaa gaagaactcc aggattgcaa aatttctcct 600

ttgatggttg catacatgtt ggagagagaa ctggtccgca aaacgagatt cctcccagtg 660

gctggtggaa caagcagtgt gtacattgaa gtgttgcatt tgactcaagg aacatgctgg 720

gaacagatgt atactccagg aggggaagtg aagaatgatg atgttgatca aagcttgatt 780

attgctgcta ggaacatagt gagaagagct gcagtatcag cagacccact agcatcttta 840

ttggagatgt gccacagcac acagattggt ggaattagga tggtagacat ccttaagcag 900

aacccaacag aagagcaagc cgtggatata tgcaaggctg caatgggact gagaattagc 960

tcatccttca gttttggtgg attcacattt aagagaacaa gcggatcatc agtcaagaga 1020

gaggaagagg tgcttacggg caatcttcaa acattgaaga taagagtgca tgagggatct 1080

gaagagttca caatggttgg gagaagagca acagccatac tcagaaaagc aaccaggaga 1140

ttgattcagc tgatagtgag tgggagagac gaacagtcga ttgccgaagc aataattgtg 1200

gccatggtat tttcacaaga ggattgtatg ataaaagcag ttagaggtga tctgaatttc 1260

gtcaataggg cgaatcagcg actgaatcct atgcatcaac ttttaagaca ttttcagaag 1320

gatgcgaaag tgctttttca aaattgggga gttgaaccta tcgacaatgt gatgggaatg 1380

attgggatat tgcccgacat gactccaagc atcgagatgt caatgagagg agtgagaatc 1440

agcaaaatgg gtgtagatga gtactccagc acggagaggg tagtggtgag cattgaccgg 1500

ttcttgagag tcagggacca acgaggaaat gtactactgt ctcccgagga ggtcagtgaa 1560

acacagggaa cagagaaact gacaataact tactcatcgt caatgatgtg ggagattaat 1620

ggtcctgaat cagtgttggt caatacctat caatggatca tcagaaactg ggaaactgtt 1680

aaaattcagt ggtcccagaa ccctacaatg ctatacaata aaatggaatt tgaaccattt 1740

cagtctttag tacctaaggc cattagaggc caatacagtg ggtttgtaag aactctgttc 1800

caacaaatga gggatgtgct tgggacattt gataccgcac agataataaa acttcttccc 1860

ttcgcagccg ctccaccaga gcaaagtaga atgcagttct cctcatttac tgtgaatgtg 1920

aggggatcag gaatgagaat acttgtaagg ggcaattctc ctgtattcaa ctacaacaag 1980

gccacgaaga gactcacagt tctcggaaag gatgctggca ctttaaccga agacccagat 2040

gaaggcacag ctggagtgga gtccgctgtt ctgaggggat tcctcattct gggcaaagaa 2100

gacaggagat atgggccagc attaagcatc aatgaactga gcaaccttgc gaaaggagag 2160

aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg aaaacgggac 2220

tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280

<210> 4

<211> 2274

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggatgtca atccgacctt acttttctta aaagtgccag cacaaaatgc tataagcaca 60

actttccctt ataccggaga ccctccttac agccatggga caggaacagg atacaccatg 120

gatactgtca acaggacaca tcagtactca gaaaagggaa gatggacaac aaacaccgaa 180

actggagcac cgcaactcaa cccgattgat gggccactgc cagaagacaa tgaaccaagt 240

ggttatgccc aaacagattg tgtattggaa gcaatggctt tccttgagga atcccatcct 300

ggtatttttg aaaactcgtg tattgaaacg atggaggttg ttcagcaaac acgagtagac 360

aagctgacac aaggccgaca gacctatgac tggactttaa atagaaacca gcctgctgca 420

acagcattgg ccaacacaat agaagtgttc agatcaaatg gcctcacggc caatgagtca 480

ggaaggctca tagacttcct taaggatgta atggagtcaa tgaaaaaaga agaaatgggg 540

atcacaactc attttcagag aaagagacgg gtgagagaca atatgactaa gaaaatgata 600

acacagagaa caataggtaa aaggaaacag agattgaaca aaaggggtta tctaattaga 660

gcattgaccc tgaacacaat gaccaaagat gctgagagag ggaagctaaa acggagagca 720

attgcaaccc cagggatgca aataaggggg tttgtatact ttgttgagac actggcaagg 780

agtatatgtg agaaacttga acaatcaggg ttgccagttg gaggcaatga gaagaaagca 840

aagttggcaa atgttgtaag gaagatgatg accaattctc aggacaccga actttctttc 900

accatcactg gagataacac caaatggaac gaaaatcaga atcctcggat gtttttggcc 960

atgatcacat atatgaccag aaatcagccc gaatggttca gaaatgttct aagtattgct 1020

ccaataatgt tctcaaacaa aatggcgaga ctgggaaaag ggtatatgtt tgagagcaag 1080

agtatgaaac ttagaactca aatacctgca gaaatgctag caagcattga tttgaaatat 1140

ttcaatgatt caacaagaaa gaagattgaa aaaatccgac cgctcttaat agaggggact 1200

gcatcattga gccctggaat gatgatgggc atgttcaata tgttaagcac tgtattaggc 1260

gtctccatcc tgaatcttgg acaaaagaga tacaccaaga ctacttactg gtgggatggt 1320

cttcaatcct ctgacgattt tgctctgatt gtgaatgcac ccaatcatga agggattcaa 1380

gccggagtcg acaggtttta tcgaacctgt aagctacttg gaatcaatat gagcaagaaa 1440

aagtcttaca taaacagaac aggtacattt gaattcacaa gttttttcta tcgttatggg 1500

tttgttgcca atttcagcat ggagcttccc agttttgggg tgtctgggat caacgagtca 1560

gcggacatga gtattggagt tactgtcatc aaaaacaata tgataaacaa tgatcttggt 1620

ccagcaacag ctcaaatggc ccttcagttg ttcatcaaag attacaggta cacgtaccga 1680

tgccatagag gtgacacaca aatacaaacc cgaagatcat ttgaaataaa gaaactgtgg 1740

gagcaaaccc gttccaaagc tggactgctg gtctccgacg gaggcccaaa tttatacaac 1800

attagaaatc tccacattcc tgaagtctgc ctaaaatggg aattgatgga tgaggattac 1860

caggggcgtt tatgcaaccc actgaaccca tttgtcagcc ataaagaaat tgaatcaatg 1920

aacaatgcag tgatgatgcc agcacatggt ccagccaaaa acatggagta tgatgctgtt 1980

gcaacaacac actcctggat ccccaaaaga aatcgatcca tcttgaatac aagtcaaaga 2040

ggagtacttg aagatgaaca aatgtaccaa aggtgctgca atttatttga aaaattcttc 2100

cccagcagtt catacagaag accagtcggg atatccagta tggtggaggc tatggtttcc 2160

agagcccgaa ttgatgcacg gattgatttc gaatctggaa ggataaagaa agaagagttc 2220

actgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atag 2274

<210> 5

<211> 2151

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaagatt ttgtgcgaca atgcttcaat ccgatgattg tcgagcttgc ggaaaaaaca 60

atgaaagagt atggggagga cctgaaaatc gaaacaaaca aatttgcagc aatatgcact 120

cacttggaag tatgcttcat gtattcagat ttccacttca tcaatgagca aggcgagtca 180

ataatcgtag aacttggtga tcctaatgca cttttgaagc acagatttga aataatcgag 240

ggaagagatc gcacaatggc ctggacagta gtaaacagta tttgcaacac tacaggggct 300

gagaaaccaa agtttctacc agatttgtat gattacaagg aaaatagatt catcgaaatt 360

ggagtaacaa ggagagaagt tcacatatac tatctggaaa aggccaataa aattaaatct 420

gagaaaacac acatccacat tttctcgttc actggggaag aaatggccac aagggccgac 480

tacactctcg atgaagaaag cagggctagg atcaaaacca ggctattcac cataagacaa 540

gaaatggcca gcagaggcct ctgggattcc tttcgtcagt ccgagagagg agaagagaca 600

attgaagaaa ggtttgaaat cacaggaaca atgcgcaagc ttgccgacca aagtctcccg 660

ccgaacttct ccagccttga aaattttaga gcctatgtgg atggattcga accgaacggc 720

tacattgagg gcaagctgtc tcaaatgtcc aaagaagtaa atgctagaat tgaacctttt 780

ttgaaaacaa caccacgacc acttagactt ccgaatgggc ctccctgttc tcagcggtcc 840

aaattcctgc tgatggatgc cttaaaatta agcattgagg acccaagtca tgaaggagag 900

ggaataccgc tatatgatgc aatcaaatgc atgagaacat tctttggatg gaaggaaccc 960

aatgttgtta aaccacacga aaagggaata aatccaaatt atcttctgtc atggaagcaa 1020

gtactggcag aactgcagga cattgagaat gaggagaaaa ttccaaagac taaaaatatg 1080

aaaaaaacaa gtcagctaaa gtgggcactt ggtgagaaca tggcaccaga aaaggtagac 1140

tttgacgact gtaaagatgt aggtgatttg aagcaatatg atagtgatga accagaattg 1200

aggtcgcttg caagttggat tcagaatgag ttcaacaagg catgcgaact gacagattca 1260

agctggatag agcttgatga gattggagaa gatgtggctc caattgaaca cattgcaagc 1320

atgagaagga attatttcac atcagaggtg tctcactgca gagccacaga atacataatg 1380

aagggggtgt acatcaatac tgccttactt aatgcatctt gtgcagcaat ggatgatttc 1440

caattaattc caatgataag caagtgtaga actaaggagg gaaggcgaaa gaccaacttg 1500

tatggtttca tcataaaagg aagatcccac ttaaggaatg acaccgacgt ggtaaacttt 1560

gtgagcatgg agttttctct cactgaccca agacttgaac cacacaaatg ggagaagtac 1620

tgtgttcttg agataggaga tatgcttcta agaagtgcca taggccaggt ttcaaggccc 1680

atgttcttgt atgtgaggac aaatggaacc tcaaaaatta aaatgaaatg gggaatggag 1740

atgaggcgtt gtctcctcca gtcacttcaa caaattgaga gtatgattga agctgagtcc 1800

tctgtcaaag agaaagacat gaccaaagag ttctttgaga acaaatcaga aacatggccc 1860

attggagagt ctcccaaagg agtggaggaa agttccattg ggaaggtctg caggacttta 1920

ttagcaaagt cggtatttaa cagcttgtat gcatctccac aactagaagg attttcagct 1980

gaatcaagaa aactgcttct tatcgttcag gctcttaggg acaatctgga acctgggacc 2040

tttgatcttg gggggctata tgaagcaatt gaggagtgcc taattaatga tccctgggtt 2100

ttgcttaatg cttcttggtt caactccttc cttacacatg cattgagtta g 2151

<210> 6

<211> 1497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggcgtccc aaggcaccaa acggtcttac gaacagatgg agactgatgg agaacgccag 60

aatgccactg aaatcagagc atccgtcgga aaaatgattg gtggaattgg acgattctac 120

atccaaatgt gcacagaact taaactcagt gattatgagg gacggttgat ccaaaacagc 180

ttaacaatag agagaatggt gctctctgct tttgacgaaa ggagaaataa atacctggaa 240

gaacatccca gtgcggggaa agatcctaag aaaactggag gacctatata cagaagagta 300

aacggaaagt ggatgagaga actcatcctt tatgacaaag aagaaataag gcgaatctgg 360

cgccaagcta ataatggtga cgatgcaacg gctggtctga ctcacatgat gatctggcat 420

tccaatttga atgatgcaac ttatcagagg acaagggctc ttgttcgcac cggaatggat 480

cccaggatgt gctctctgat gcaaggttca actctcccta ggaggtctgg agccgcaggt 540

gctgcagtca aaggagttgg aacaatggtg atggaattgg tcaggatgat caaacgtggg 600

atcaatgatc ggaacttctg gaggggtgag aatggacgaa aaacaagaat tgcttatgaa 660

agaatgtgca acattctcaa agggaaattt caaactgctg cacaaaaagc aatgatggat 720

caagtgagag agagccggaa cccagggaat gctgagttcg aagatctcac ttttctagca 780

cggtctgcac tcatattgag agggtcggtt gctcacaagt cctgcctgcc tgcctgtgtg 840

tatggacctg ccgtagccag tgggtacgac tttgaaagag agggatactc tctagtcgga 900

atagaccctt tcagactgct tcaaaacagc caagtgtaca gcctaatcag accaaatgag 960

aatccagcac acaagagtca actggtgtgg atggcatgcc attctgccgc atttgaagat 1020

ctaagagtat tgagcttcat caaagggacg aaggtggtcc caagagggaa gctttccact 1080

agaggagttc aaattgcttc caatgaaaat atggagacta tggaatcaag tacacttgaa 1140

ctgagaagca ggtactgggc cataaggacc agaagtggag gaaacaccaa tcaacagagg 1200

gcatctgcgg gccaaatcag catacaacct acgttctcag tacagagaaa tctccctttt 1260

gacagaacaa ccgttatggc agcattcact gggaatacag aggggagaac atctgacatg 1320

aggaccgaaa tcataaggat gatggaaagt gcaagaccag aagatgtgtc tttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcagcgagcc cgatcgtgcc ttcctttgac 1440

atgagtaatg aaggatctta tttcttcgga gacaatgcag aggagtacga caattaa 1497

<210> 7

<211> 982

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccgtc aggccccctc 60

aaagccgaga tcgcacagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120

gttctcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa ggggatttta 180

ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240

caaaatgccc ttaatgggaa cggggatcca aataacatgg acaaagcagt taaactgtat 300

aggaagctca agagggagat aacattccat ggggccaaag aaatctcact cagttattct 360

gctggtgcac ttgccagttg tatgggcctc atatacaaca ggatgggggc tgtgaccact 420

gaagtggcat ttggcctggt atgtgcaacc tgtgaacaga ttgctgactc ccagcatcgg 480

tctcataggc aaatggtgac aacaaccaac ccactaatca gacatgagaa cagaatggtt 540

ttagccagca ctacagctaa ggctatggag caaatggctg gatcgagtga gcaagcagca 600

gaggccatgg aggttgctag tcaggctagg caaatggtgc aagcgatgag aaccattggg 660

actcatccta gctccagtgc tggtctgaaa aatgatcttc ttgaaaattt gcaggcctat 720

cagaaacgaa tgggggtgca gatgcaacgg ttcaagtgat cctctcgcta ttgccgcaaa 780

tatcattggg atcttgcact tgatattgtg gattcttgat cgtctttttt tcaaatgcat 840

ttaccgtcgc tttaaatacg gactgaaagg agggccttct acggaaggag tgccaaagtc 900

tatgagggaa gaatatcgaa aggaacagca gagtgctgtg gatgctgacg atggtcattt 960

tgtcagcata gagctggagt aa 982

<210> 8

<211> 838

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa 60

cgagttgtag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag 120

aaatccctaa gaggaagggg cagcactctt ggtctggaca tcgagacagc cacacgtgct 180

ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc 240

atggcctctg tacctgcgtc gcgttaccta accgacatga ctcttgagga aatgtcaagg 300

gaatggtcca tgctcatacc caagcagaaa gtggcaggcc ctctttgtat cagaatggac 360

caggcgatca tggataaaaa catcatactg aaagcgaact tcagtgtgat ttttgaccgg 420

ctggagactc taatattgct aagggctttc accgaagagg gagcaattgt tggcgaaatt 480

tcaccattgc cttctcttcc aggacatact gctgaggatg tcaaaaatgc agttggagtc 540

ctcatcggag gacttgaatg gaatgataac acagttcgag tctctgaaac tctacagaga 600

ttcgcttgga gaagcagtaa tgagaatggg agacctccac tcactccaaa acagaaacga 660

gaaatggcgg gaacaattag gtcagaagtt tgaagaaata agatggttga ttgaagaagt 720

gagacacaaa ctgaaggtaa cagagaatag ttttgagcaa ataacattta tgcaagcctt 780

acatctattg cttgaagtgg agcaagagat aagaactttc tcatttcagc ttatttaa 838

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggagaaca tagtacttct tcttg 25

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gccccgaaca ggcctctagt ttctcttaga ggactatttc tgagc 45

<210> 11

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

actagaggcc tgttcggggc gatagcaggg t 31

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttaaatgcaa attctgcact gtaac 25

Claims

1. The artificially recombined H5N8 influenza virus strain H5-Re14 is preserved in China center for type culture Collection, is addressed to Wuhan university in Wuhan, China, and has the preservation number of CCTCC NO: v202160, preservation time 2021, 8 months and 3 days.

2. The strain H5-Re14 as claimed in claim 1, and application thereof in preparing medicines for preventing diseases caused by H5N8 influenza virus.

3. The use according to claim 2, wherein the influenza virus H5N8 is the A/swan/Shanxi/4-1/2020(H5N8) strain.

4. A vaccine comprising the strain H5-Re14 of claim 1.

5. The strain H5-Re14 of claim 1, wherein the strain H5-Re14 comprises:

(1) designing HA gene specific amplification and mutation primers, amplifying HA1 and HA2 segments of HA genes of a highly pathogenic H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) strain by using an RT-PCR method, and mutating amino acid at a cracking site of the HA genes from-REKRRKR-to-RETR-while amplifying the HA1 segment, so that the molecular characteristics of the highly pathogenic avian influenza virus are mutated into the molecular characteristics of a typical low pathogenic avian influenza virus; then, an HA segment with a cracking site being the molecular characteristic of the typical low-pathogenicity avian influenza virus is amplified by utilizing an overlap extension reaction, and a pBD plasmid is inserted to construct a recombinant HA gene bidirectional transcription vector;

(2) amplifying the NA gene full-length segment of H5N8 subtype avian influenza virus A/swan/Shanxi/4-1/2020(H5N8) by using an RT-PCR method, inserting pBD plasmid, and constructing a recombinant NA gene bidirectional transcription vector;

(3) mixing 2 recombinant pBD bidirectional transcription vector plasmids of HA and NA genes constructed in the steps (1) and (2) with 6 pBD bidirectional transcription vector plasmids of PB2, PB1, PA, NP, M and NS genes of an influenza virus chick embryo high-titer adaptive strain A/PR/8/34(H1N1), inoculating 293T cells with a transfection reagent, and inoculating the cells and supernatant harvested after culture into chick embryos to obtain the H5N8 avian influenza recombinant virus strain H5-Re14 with HA activity.

6. The preparation method of claim 5, wherein the primer pair for amplifying the HA1 gene is shown as SEQ ID No.9 and SEQ ID No.10, wherein SEQ ID No.10 is a mutation primer of HA cleavage site; the primer pair for amplifying the HA2 gene is shown as SEQ ID No.11 and SEQ ID No. 12.