A recombinant turkey herpes virus strain expressing H9 subtype AIV HA gene and IBV S gene and its application

Abstract

The present invention discloses a recombinant turkey herpes virus strain expressing H9 subtype AIV HA gene and IBV S gene and its application, belonging to the technical field of medicine or veterinary medicine. The recombinant turkey herpes virus strain is obtained by inserting an expression frame comprising a chicken β-actin promoter, AIV HA gene and IBV S gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylic acid sequence into the HVT genome. The recombinant virus strain obtained by the present invention has good in vitro replication ability and genetic stability. After immunizing SPF chickens, the SPF chickens can obtain immune protection against H9 subtype AIV and QX type IBV. The present invention provides a new technical means for preventing H9 subtype avian influenza and chicken infectious bronchitis at the same time.

Description

Technical Field

[0001] The present invention relates to a recombinant turkey herpes virus strain expressing the HA gene of H9 subtype avian influenza virus and the S gene of infectious bronchitis virus and its application in preparing H9 subtype avian influenza and chicken infectious bronchitis vaccines. The present invention belongs to the technical field of medicine or veterinary medicine. Background Art

[0002] The H9 subtype avian influenza virus (AIV) was first isolated from a turkey flock in the United States in 1966. It is now distributed worldwide. Its widespread prevalence in poultry poses a continuous threat to the global poultry industry and public health security. Since the H9 subtype AIV was first isolated in Guangdong Province in 1992, the disease has been prevalent in my country for more than 30 years. Broilers are the main host of the H9 subtype AIV. In recent years, the virus has continued to maintain a high level of prevalence in my country and has become one of the most serious pathogens affecting the development of my country's broiler farming industry. In particular, when mixed with pathogens such as infectious bronchitis virus (IBV), broilers will suffer from severe bronchial obstruction symptoms, which seriously affects the health of the flock. AIV belongs to the Orthomyxoviridae family. The HA protein it encodes can recognize specific receptors and has strong immunogenicity. It is the main host protective antigen of the virus and can induce the production of neutralizing antibodies. The H9 subtype avian influenza is currently mainly prevented and controlled through inactivated vaccines. However, in recent years, the isolation rate of H9 subtype AIV in my country has increased year by year, indicating that traditional inactivated vaccine immunization can no longer effectively prevent and control the virus infection. There is an urgent need to develop a safer and more efficient new vaccine.

[0003] Infectious Bronchitis (IB) is an acute, highly contagious disease caused by IBV, which can cause respiratory, urinary and reproductive system infections in chickens. The disease is prevalent throughout the year, and is particularly severe in winter and spring. Clinically, it is often mixed with H9 subtype AIV, Escherichia coli and mycoplasma, resulting in high morbidity and mortality. Chickens of different ages and strains can be infected and fall ill, and the incidence rate of chicks is high. It is a disease that is difficult to prevent and control. The spike protein (S) is the most important structural protein of IBV, which can induce the production of neutralizing antibodies and is the main host protective antigen of IBV. The S protein consists of two subunits, S1 and S2, both of which are glycoproteins. The S1 protein plays a decisive role in the virulence, tissue tropism and serotype of the virus, and can induce humoral and cellular immune responses in the host. At present, the dominant genotype of IBV in my country is the genotype I group 19 subtype (QX type), which is mainly controlled by traditional attenuated live vaccines and inactivated vaccines.

[0004] Herpesvirus of turkey (HVT) is a herpesvirus that is non-pathogenic to chickens and is widely used in the prevention of Marek's disease in chickens at home and abroad. As a herpesvirus, HVT has a large genome and many replication-non-essential genes that can be inserted or replaced by exogenous genes. It is an ideal viral vector for constructing recombinant live vector vaccines. Compared with other viral vectors, HVT has many advantages as a viral vector. It is non-pathogenic to chickens and other animals and is safe to use; the immune effect of HVT vaccine is not interfered by maternal antibodies and can be used for early immunization of one-day-old chicks in hatcheries; after vaccination, the virus exists in the chicken for a long time, stimulating the body to produce high antibody levels and maintain them for life, and lifelong immunity can be obtained with one vaccination; HVT vaccine not only has low production costs, but also can be freeze-dried, easy to store and transport. Summary of the invention

[0005] The present invention aims to provide a recombinant turkey herpes virus strain expressing the HA gene of H9 subtype avian influenza virus and the S gene of infectious bronchitis virus and its application in preparing H9 subtype avian influenza and chicken infectious bronchitis vaccines.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The recombinant turkey herpesvirus strain for co-expressing the avian influenza virus HA gene and the infectious bronchitis virus S gene provided by the present invention is obtained by inserting the expression framework CAGW-HA2AS comprising a chicken β-actin promoter, AIV HA gene and IBV S gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence into the turkey herpesvirus genome.

[0008] Among them, preferably, the expression frame CAGW-HA2AS containing chicken β-actin promoter, AIV HA gene and IBV S gene coding sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence is inserted into the HVT genome between nucleotides 95322-95323 or 112071-112088.

[0009] Among them, preferably, the AIV is an H9 subtype strain, and the IBV is a QX subtype strain.

[0010] Preferably, the AIV HA gene is obtained by optimizing the chicken codon of the coding region of the H9 subtype AIV HA gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0011] Among them, preferably, the IBV S gene is obtained by mutating the S1-S2 cleavage site in the QX type IBV S protein sequence from RRRR to GSAS, mutating the amino acids at positions 695, 768, 775, 817, 862 and 863 to proline, and replacing the amino acids at positions 1097-1165 at the C-terminus with the T4 fiber protein trimer domain, and optimizing the chicken codon design, and its nucleotide sequence is shown in SEQ ID NO.2.

[0012] Among them, preferably, the AIV HA gene and IBV S gene coding sequences are obtained by connecting the HA gene coding sequence and the S gene coding sequence with the porcine teschovirus 2A (P2A) self-cleavage peptide coding sequence, and the nucleotide sequence thereof is shown in SEQ ID NO.3.

[0013] Furthermore, the present invention also proposes a method for constructing the recombinant turkey herpes virus strain, comprising the following steps:

[0014] (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0015] Extract the genomic DNA of HVT FC126 strain, and clone the genomic DNA of FC126 strain into pCC1Fos vector in segments; select 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that clone the genomic DNA fragments of FC126 strain and can be spliced ​​to cover the complete HVT genome; among them, HVT01 contains the nucleotide fragment of 1-30189 of FC126 genome, HVT02 contains the nucleotide fragment of 21572-61476 of FC126 genome, HVT03 contains the nucleotide fragment of 50871-90478 of FC126 genome, HVT04 contains the nucleotide fragment of 73762-108139 of FC126 genome, HVT05 contains the nucleotide fragment of 96776-135815 of FC126 genome, and HVT06 contains the nucleotide fragment of 129602-159160 of FC126 genome;

[0016] (2) Construction of recombinant cosmids expressing HA and S genes

[0017] A recombinant plasmid comprising a chicken β-actin promoter, AIV HA gene and IBV S gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylation sequence expression frame CAGW-HA2AS is constructed, wherein the HA gene and S gene coding regions are connected by a porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence; the expression frame CAGW-HA2AS is cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 or the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 by using the Red / ET recombination method, and a recombinant cosmid that co-expresses the HA gene and the S gene is constructed and named HVT04-53-HA2AS or HVT05-65-HA2AS;

[0018] (3) Rescue of recombinant HVT expressing HA and S genes

[0019] The recombinant cosmids HVT04-53-HA2AS, HVT05-65-HA2AS and the other five parental cosmids HVT01, HVT02, HVT03, HVT04, HVT05 and HVT06 cloned with the genomic fragment of HVT FC126 strain were extracted using a plasmid extraction kit; the above recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method, the viruses were harvested after the appearance of plaque lesions in culture, and were serially passaged and preserved in CEF cells to rescue the recombinant viruses with the expression framework HA2AS inserted between nucleotides 95322-95323 or 112071-112088 of the HVT genome, which were named H20301 or H20302, respectively.

[0020] Preferably, the HVT strain is HVT FC126 strain, and the GenBank accession number of the genome sequence is AF291866.

[0021] Among them, preferably, the nucleotide sequence of the expression framework CAGW-HA2AS is shown in SEQ ID NO.4.

[0022] Furthermore, the present invention also proposes the use of the recombinant turkey herpes virus strain in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza and chicken infectious bronchitis.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention utilizes recombinant cloning technology to insert an expression framework containing the H9 subtype AIV HA gene and the IBV S gene into the 95322-95323 position or the 112071-112088 position nucleotides of the HVT FC126 strain genome, and constructs recombinant HVT virus strains H20301 and H20302 that co-express the HA gene and the S gene. Experiments have shown that the recombinant virus strains H20301 and H20302 have good in vitro replication characteristics and genetic stability. After immunizing specific pathogen-free (SPF) chickens, the SPF chickens can obtain immune protection against the H9 subtype AIV and QX type IBV strong toxins, and can be used for the preparation of H9 subtype avian influenza and chicken infectious bronchitis vaccines. The invention provides a new technical means for preventing H9 subtype avian influenza and chicken infectious bronchitis at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The PCR identification results of the recombinant cosmids HVT04-53-HA2AS and HVT05-65-HA2AS expressing the HA gene and S gene;

[0026] Figure 2 The plaque lesions produced by recombinant viruses H20301 and H20302 on CEF;

[0027] Figure 3 The results of PCR identification of the genomic DNA of the recombinant viruses H20301 and H20302;

[0028] Figure 4 Indirect immunofluorescence assay was used to detect the expression of HA and S proteins in CEFs infected with recombinant virus H20301 or H20302;

[0029] Figure 5 The replication kinetics curves of recombinant viruses H20301 and H20302 on CEF cells;

[0030] Figure 6 The results of PCR test for genetic stability of recombinant viruses H20301 and H20302;

[0031] Figure 7 The results of indirect immunofluorescence test for the expression of HA and S proteins by the 20th generation recombinant viruses H20301 and H20302;

[0032] Figure 8 The results are as follows: The results of the test of the titer of H9 subtype AIV HI antibody induced by recombinant viruses H20301 and H20302 in SPF chickens 4 weeks after inoculation;

[0033] Fig. 9These are the results of the test on the IBV neutralizing antibody titer induced by recombinant viruses H20301 and H20302 inoculated SPF chickens 4 weeks after inoculation. DETAILED DESCRIPTION

[0034] The present invention is further described below. The implementation cases introduced in this description are only exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, the details and forms of the technical solution of the present invention may be partially modified or replaced, but such modification or replacement is within the scope of protection of the present invention.

[0035] Example 1: Construction and identification of recombinant HVT expressing H9 subtype AIV HA gene and IBV S gene

[0036] 1.1 Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0037] Extract HVT FC126 strain genomic DNA, the HVT FC126 strain genomic DNA sequence GenBank accession number is AF291866. According to the instructions of Fosmid Library Production Kit, the genomic DNA of FC126 strain was cloned into pCC1Fos vector. According to the results of terminal sequencing of recombinant cosmids, six recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 were selected, which cloned the genomic DNA fragments of FC126 strain and could be spliced ​​to cover the complete HVT genome. Among them, HVT01 contains the nucleotide fragment of 1-30189 of the FC126 genome, HVT02 contains the nucleotide fragment of 21572-61476 of the FC126 genome, HVT03 contains the nucleotide fragment of 50871-90478 of the FC126 genome, HVT04 contains the nucleotide fragment of 73762-108139 of the FC126 genome, HVT05 contains the nucleotide fragment of 96776-135815 of the FC126 genome, and HVT06 contains the nucleotide fragment of 129602-159160 of the FC126 genome. The above 6 recombinant cosmids cloned with FC126 genomic DNA fragments were extracted, and the 6 recombinant cosmids were co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. The appearance of cytopathic effect can be observed 4-5 days after transfection, that is, the parental virus strain FC126 was rescued.

[0038] 1.2 Construction and identification of recombinant cosmids expressing HA and S genes

[0039] According to the HA gene coding region sequence of the H9N2 subtype AIV TJ strain isolated and identified in our laboratory, the target gene H9HA (shown in SEQ ID NO.1) was synthesized after chicken codon optimization, and the porcine Teschovirus 2A (P2A) self-cleavage peptide sequence was added to its C-terminus. According to the S protein sequence of the QX type IBV SX strain (GenBank accession number OQ189490), its S1-S2 cleavage site RRRR was mutated to GSAS, and the amino acids at positions 695, 768, 775, 817, 862 and 863 were mutated to proline, and the amino acids at positions 1097-1165 at its C-terminus were replaced with the T4 fiber protein trimer domain, and the target gene IBVS6P (shown in SEQ ID NO.2) was synthesized after chicken codon optimization. The target gene HA2AS (shown in SEQ ID NO.3) containing the coding sequences of HA and S genes was obtained by fusion PCR amplification using primers H9P1F, H9S6P1R, H9S6P2F and S6P2R (Table 1), wherein the coding regions of HA gene and S gene were connected by self-cleaving peptide P2A. The target gene PCR product was purified and cloned into the downstream of chicken β-actin promoter of pCAGGS vector; at the same time, in order to enhance the transcription and translation efficiency of the target gene, the woodchuck hepatitis virus post-transcriptional regulatory sequence (GenBank: MQ208857.1) was inserted between the target gene HA2AS and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-HA2AS that co-expresses H9 AIV HA gene and IBV S gene, wherein the HA2AS gene expression framework (named CAGW-HA2AS) sequentially comprises: chicken β-actin promoter-target gene HA2AS-woodchuck hepatitis virus post-transcriptional regulatory sequence-rabbit β-globulin polyadenylation sequence. The recombinant plasmid was sequenced using primers CAGF and CAGR (Table 1), and the nucleotide sequence of the target gene HA2AS was obtained as shown in SEQ ID NO.3, which was consistent with the expectation, indicating that the recombinant plasmid was constructed correctly.

[0040] According to the instructions of the Counter Selection BAC Modification Kit, the HA2AS gene expression framework CAGW-HA2AS (shown in SEQ ID NO.4) was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 using the Red / ET recombination method to construct the recombinant cosmid HVT04-53-HA2AS that co-expresses the HA gene and the S gene. The target gene-specific primer IBVS2046F and the target gene downstream homology arm primer HVT54R (Table 1) were used to identify whether the recombinant cosmid HVT04-53-HA2AS was inserted into the target gene expression framework by PCR. The results showed that the PCR fragment of about 2700 bp was obtained ( Figure 1 ), which was consistent with expectations; sequencing results showed that the PCR product contained the S gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parental cosmid HVT04 had no target gene insertion sequence, and the PCR result was negative. The above results indicate that the recombinant cosmid HVT04-53-HA2AS was constructed correctly.

[0041] According to the instructions of the Counter Selection BAC Modification Kit, the HA2AS gene expression framework CAGW-HA2AS (SEQ ID NO.4) was inserted into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 using the Red / ET recombination method to construct the recombinant cosmid HVT05-65-HA2AS that co-expresses the HA gene and the S gene. The target gene-specific primer IBVS2046F and the target gene downstream homology arm primer HVT65F (Table 1) were used to identify whether the recombinant cosmid HVT05-65-HA2AS was inserted into the target gene expression framework by PCR. The results showed that the obtained PCR fragment was about 2700 bp ( Figure 1 ), which was consistent with expectations; sequencing results showed that the PCR product contained the S gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parental cosmid HVT05 had no target gene insertion sequence, and the PCR result was negative. The above results indicate that the recombinant cosmid HVT05-65-HA2AS was constructed correctly.

[0042]

[0043] 1.3 Rescue and identification of recombinant HVT expressing HA and S genes

[0044] Recombinant cosmids HVT04-53-HA2AS, HVT05-65-HA2AS and other parental cosmids HVT01, HVT02, HVT03, HVT04, HVT05, HVT06 cloned with HVT genome fragments were extracted using a plasmid extraction kit. CEF cells were co-transfected with recombinant cosmids HVT04-53-HA2AS and parental cosmids HVT01, HVT02, HVT03, HVT05, HVT06 using the calcium phosphate transfection method, and the virus was harvested after plaque lesions appeared after 4-5 days of culture, and was continuously passaged and preserved in CEF cells to rescue the recombinant virus H20301 with the HA2AS expression frame inserted between nucleotides 95322-95323 of the HVT genome. The recombinant cosmid HVT05-65-HA2AS was co-transfected with the parental cosmids HVT01, HVT02, HVT03, HVT04, and HVT06 into CEF cells. After 4-5 days of culture, the virus was harvested after plaque lesions appeared. The virus was continuously passaged and stored in CEF cells to rescue the recombinant virus H20302 in which the HA2AS expression framework was inserted between nucleotides 112071-112088 of the HVT genome. The plaque lesions produced by the recombinant viruses H20301 and H20302 on CEF are shown in Figure 2. Figure 2 shown.

[0045] The genomic DNA of the fifth-generation recombinant viruses H20301 and H20302 was extracted, and the genomic DNA of the parental virus FC126 was set as a control. The recombinant viruses were identified by PCR using the target gene-specific primer IBVS2046F and the target gene downstream homology arm primer HVT54R or HVT65F (Table 1), and the PCR products were further sequenced and analyzed. The PCR results showed that the PCR product obtained by recombinant virus amplification was about 2700 bp in length, which was consistent with the expected size ( Figure 3 ). The sequencing results showed that the PCR product contained the S gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parent virus FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene HA2AS was correctly inserted into the genome of the HVT FC126 strain, and the recombinant viruses H20301 and H20302 were correctly constructed.

[0046] Example 2: In vitro biological characteristics analysis of recombinant HVT expressing H9 subtype AIV HA gene and IBV S gene

[0047] 2.1 Detection of HA and S proteins expressed by recombinant viruses H20301 and H20302

[0048] The recombinant viruses H20301, H20302 and the parental virus FC126 were inoculated into CEF cells, and after plaque lesions appeared after 3-4 days of culture, the cells were fixed with anhydrous ethanol. The HA protein polyclonal antibody and IBV positive serum were used as primary antibodies, and FITC-labeled goat anti-rabbit IgG and FITC-labeled rabbit anti-chicken IgG were used as secondary antibodies. The expression of HA protein and S protein was detected by indirect immunofluorescence test, and CEF infected with the parental virus FC126 was used as a negative control. The results showed that the cells infected with the recombinant viruses H20301 and H20302 were able to react with the HA protein polyclonal antibody and IBV positive serum, showing green fluorescence signals ( Figure 4 ). No fluorescence was observed in cells infected with the parental virus FC126 strain and in control cells that were not infected. The above results indicate that the recombinant viruses H20301 and H20302 can co-express HA protein and S protein in infected cells.

[0049] 2.2 Analysis of in vitro replication characteristics of recombinant viruses H20301 and H20302

[0050] The recombinant viruses H20301, H20302 and the parental virus FC126 were inoculated on CEF in a 6-well plate at a dose of 100 plaque-forming units (PFU). The virus-containing cells were collected every 24 hours after infection until 144 hours after infection. The viruses collected at each time point were inoculated into CEF, the number of plaques in the virus solution at each time point was determined, and the in vitro replication kinetic curve of the virus was drawn to analyze the in vitro replication characteristics of the recombinant viruses H20301, H20302 and the parental virus FC126 in CEF. The results showed that the replication titers of the recombinant viruses H20301, H20302 and the parental virus FC126 reached the highest peak 120 hours after infection, which were 2.11×10 5 PFU / ml, 1.92×10 5 PFU / ml and 2.02×10 5 PFU / ml, the titer of the recombinant virus at each time point after infection with CEF was not significantly different from that of the parental virus ( P >0.05) ( Figure 5 ). The above results show that the recombinant viruses H20301 and H20302 have good replication ability on CEF, and their in vitro replication characteristics are consistent with those of the parent virus FC126 strain.

[0051] 2.3 Genetic stability testing of recombinant viruses H20301 and H20302

[0052] The 5th generation recombinant viruses H20301 and H20302 were respectively propagated on CEF for 20 generations. The 10th, 15th, and 20th generation cytotoxic cells were selected, and the viral genomic DNA was extracted. The target gene-specific primer IBVS2046F and the downstream homology arm primer HVT54R or HVT65F were used for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the recombinant virus genome. At the same time, the above-mentioned 20th generation recombinant virus was inoculated into CEF cells, and the expression stability of the target genes HA and S was detected by indirect immunofluorescence test using HA protein polyclonal antibodies and IBV positive serum.

[0053] PCR identification results showed that the target genes HA and S were stably present in the HVT genome, and the target gene bands of about 2700 bp could be amplified in the 10th, 15th, and 20th generation recombinant viruses ( Figure 6 ), which was consistent with expectations. The 20th generation recombinant virus genomic DNA was amplified using primers CAGF and CAGR and the PCR products were sequenced, and it was found that the target gene sequence inserted into the HVT genome was correct. The above 20th generation recombinant virus was taken for indirect immunofluorescence test to detect the expression of the target genes HA and S during the passage process. The results showed that after the recombinant viruses H20301 and H20302 were continuously passed on CEF cells to the 20th generation, the target genes HA and S could still be stably expressed ( Figure 7 ). The above results show that the recombinant viruses H20301 and H20302 have good genetic stability.

[0054] Example 3: Safety and immunogenicity testing of recombinant viruses H20301 and H20302

[0055] 3.1 Safety testing of recombinant viruses H20301 and H20302

[0056] The fifth-generation recombinant virus H20301 or H20302 was inoculated into one-day-old SPF chickens at a dose of 4000 PFU / chicken, and the clinical symptoms of each group of chickens were observed every day after inoculation. 28 days after inoculation, 5 test chickens were randomly selected from each group, and their body weights were weighed to evaluate the effects of recombinant viruses H20301 and H20302 on the growth and development of test chickens; 5 chickens selected from each group were killed, and organs such as the bursa of Fabricius, thymus, spleen, and liver were collected and weighed to observe whether there were symptoms of atrophy or swelling. The results showed that the recombinant virus H20301 or H20302 did not cause adverse clinical reactions in the test chickens after inoculation of SPF chickens, and the feeding and drinking of the immunized chickens were normal. 28 days after inoculation, the weighing and autopsy results showed that there was no significant difference in the body weight of the chickens in the inoculation groups of the recombinant viruses H20301 and H20302 and the normal uninoculated control group. The chickens were dissected and the bursa of Fabricius, thymus, spleen, liver and other organs were collected. The results showed that the above organs were normal and no obvious clinical lesions were found. The above results show that the recombinant viruses H20301 and H20302 are safe for SPF chickens.

[0057] 3.2 Immunoprotective test of recombinant viruses H20301 and H20302 against H9 subtype AIV

[0058] Forty one-day-old SPF chicks were randomly divided into four groups, with 10 chicks in each group. Group 1 was subcutaneously inoculated with the recombinant virus H20301 at a dose of 4000 PFU / chicken, Group 2 was subcutaneously inoculated with the recombinant virus H20302 at a dose of 4000 PFU / chicken, Group 3 was inoculated with the parental virus FC126 at the same dose, and Group 4 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the HI antibody titer of chickens in each group was detected by hemagglutination inhibition test. 28 days after immunization, groups 1-3 were nasally challenged with the H9N2 subtype AIV TJ strain; Group 4 was not challenged and served as a healthy control group. After the challenge, the chickens were observed for 14 days and the clinical symptoms of the test chickens were recorded. Five days after the challenge, laryngeal and cloacal swabs were collected from each chicken in each group for virus isolation. The collected cotton swabs were repeatedly frozen and thawed three times and then centrifuged. The supernatant was inoculated into 10-day-old SPF chicken embryos through the allantoic cavity. The chicken embryos were placed in an incubator at 37°C for 96 hours, and then the allantoic fluid was collected and the HA titer was determined. A titer of no less than 1:16 was considered positive for virus isolation. The samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0059] The results showed that 28 days after the recombinant virus H20301 or H20302 was inoculated into one-day-old SPF chicks, the average titer of HI antibody in the test chickens could reach 1:338 or 1:446, respectively, while the HI antibody titer in the parental virus FC126 inoculation group was negative ( Figure 8). The above results show that the recombinant virus H20301 or H20302 induced a good immune response in chicks after inoculation. After the experimental chickens were challenged with H9 subtype AIV, 9 out of 10 chickens in the parental virus FC126 strain inoculation group were positive for virus isolation, and the virus isolation of the experimental chickens in the blank control group without infection was negative, and the infection test was established. After the recombinant virus H20301 or H20302 was inoculated with chickens to challenge H9 subtype AIV, during the observation period, all chickens had normal feeding, drinking, mental state, movement, etc., without obvious adverse clinical symptoms, virus isolation was negative, and the infection protection rate was 100%.

[0060] 3.3 Immunoprotective test of recombinant viruses H20301 and H20302 against virulent IBV

[0061] Forty one-day-old SPF chicks were randomly divided into four groups, with 10 chicks in each group. Group 1 was subcutaneously inoculated with the recombinant virus H20301 at a dose of 4000 PFU / chicken, Group 2 was subcutaneously inoculated with the recombinant virus H20302 at a dose of 4000 PFU / chicken, Group 3 was inoculated with the parental virus FC126 at the same dose, and Group 4 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the titer of IBV neutralizing antibodies was detected. 28 days after immunization, Groups 1-3 were challenged with the QX-type virulent SD strain by nose drops and eye drops; Group 4 was not challenged and served as a healthy control group. After 14 days of observation, the clinical symptoms and deaths of the experimental chickens in each group were recorded; 14 days after the challenge, all experimental chickens were killed and dissected, and the lesions of organs such as the trachea, larynx and kidneys were observed and recorded.

[0062] The results showed that 28 days after the recombinant virus H20301 or H20302 was inoculated into one-day-old SPF chicks, the average titer of IBV neutralizing antibody in the experimental chickens could reach 1:104 or 1:128, respectively, while the titer of IBV neutralizing antibody in the parent virus FC126 inoculation group was negative ( Fig. 9). The above results show that the recombinant virus H20301 or H20302 induced a good immune response in chicks after inoculation. After the parental virus FC126 strain inoculation group was challenged with IBV, 9 of the 10 chickens became ill, of which 2 died. The sick chickens showed clinical symptoms such as depression, runny nose, and messy feathers. Necropsy showed kidney enlargement, spotted kidney, laryngeal or tracheal bleeding, tracheal mucus and other lesions; the test chickens in the blank control group without challenge showed no abnormal clinical and autopsy symptoms, and the challenge test was established. After the recombinant virus H20301 inoculation group was challenged with QX type IBV, all the test chickens survived during the observation period; 9 of the chickens had normal feeding, drinking, mental state, and movement, without obvious adverse clinical symptoms and autopsy lesions; 1 chicken showed symptoms of depression, and autopsy showed mucus in the larynx. After the 10 chickens in the recombinant virus H20302 inoculation group were challenged with the strong QX-type IBV, all the test chickens survived during the observation period, and their feeding, drinking, mental state, and movement were normal, with no obvious adverse clinical symptoms or autopsy lesions. The above results show that the protection rates of the recombinant viruses H20301 and H20302 against the strong QX-type IBV were 90% and 100%, respectively.

Claims

1. A recombinant turkey herpesvirus strain (Herpesvirus of turkey, HVT) that co-expresses the HA gene of avian influenza virus (Avian influenza virus, AIV) and the S gene of infectious bronchitis virus (Infectious Bronchitis virus, IBV), characterized in that: The recombinant turkey herpesvirus strain is obtained by inserting the expression frame CAGW-HA2AS containing chicken β-actin promoter, AIV HA gene and IBV S gene coding sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence into the 95322-95323 position or the 112071-112088 position nucleotides of the HVT genome; Among them, the AIV HA gene and IBV S gene coding sequences are obtained by connecting the HA gene coding sequence and the S gene coding sequence with the porcine teschovirus 2A (P2A) self-cleavage peptide coding sequence; the IBV S gene is obtained by mutating the S1-S2 cleavage site in the QX type IBV S protein sequence from RRRR to GSAS, and mutating its 695th, 768th, 775th, 817th, 862nd and 863rd amino acids to proline, and replacing its C-terminal 1097-1165 amino acids with the T4 fiber protein trimer domain, and after chicken codon optimization design, its nucleotide sequence is shown in SEQ ID NO.

2.

2. The recombinant turkey herpes virus strain according to claim 1, characterized in that The AIV is a H9 subtype strain, and the IBV is a QX subtype strain.

3. The recombinant turkey herpes virus strain according to claim 1, characterized in that The AIV HA gene is obtained by optimizing the chicken codon of the H9 subtype AIV HA gene coding region, and its nucleotide sequence is shown in SEQ ID NO.

1.

4. The recombinant turkey herpes virus strain according to claim 1, characterized in that The nucleotide sequences of the AIV HA gene and IBV S gene coding sequences are shown in SEQ ID NO.

3.

5. A method for constructing the recombinant turkey herpes virus strain according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain Extract the genomic DNA of HVT FC126 strain, clone the genomic DNA of FC126 strain into pCC1Fos vector, and select 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that clone the genomic DNA fragments of FC126 strain and can be spliced ​​to cover the complete HVT genome; among them, HVT01 contains the nucleotide fragment of 1-30189 of FC126 genome, HVT02 contains the nucleotide fragment of 21572-61476 of FC126 genome, HVT03 contains the nucleotide fragment of 50871-90478 of FC126 genome, HVT04 contains the nucleotide fragment of 73762-108139 of FC126 genome, HVT05 contains the nucleotide fragment of 96776-135815 of FC126 genome, and HVT06 contains the nucleotide fragment of 129602-159160 of FC126 genome; (2) Construction of recombinant cosmids expressing HA and S genes A recombinant plasmid comprising a chicken β-actin promoter, AIV HA gene and IBV S gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylation sequence expression framework CAGW-HA2AS is constructed, wherein the HA gene and S gene coding regions are connected by a porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence; the expression framework CAGW-HA2AS is cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 or the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 by using the Red / ET recombination method, and a recombinant cosmid that co-expresses the HA gene and the S gene is constructed and named HVT04-53-HA2AS or HVT05-65-HA2AS; (3) Rescue of recombinant HVT expressing HA and S genes The recombinant cosmids HVT04-53-HA2AS, HVT05-65-HA2AS and the parental cosmids HVT01, HVT02, HVT03, HVT04, HVT05 and HVT06 cloned with the genome fragment of HVTFC126 strain were extracted using a plasmid extraction kit; the above-mentioned recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method, the viruses were harvested after the appearance of plaque lesions in culture, and were continuously passaged and preserved in CEF cells to rescue the recombinant viruses in which the expression framework CAGW-HA2AS was inserted between nucleotides 95322-95323 or 112071-112088 of the HVT FC126 strain genome, which were named H20301 or H20302, respectively.

6. The method according to claim 5, characterized in that The HVT strain is HVT FC126 strain, and the GenBank accession number of its genome sequence is AF291866.

7. The method according to claim 5, characterized in that The nucleotide sequence of the expression framework CAGW-HA2AS is shown in SEQ ID NO.

4.

8. Use of the recombinant turkey herpesvirus strain according to any one of claims 1 to 4 in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza and infectious bronchitis.

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