Recombinant herpesvirus turkey strain for co-expressing H9 subtype AIV HA gene, IBDV VP2 gene and ILTV gB, gD and gI genes and application

Abstract

The invention discloses a recombinant herpesvirus turkey strain for co-expressing an H9 subtype AIVHA gene, an IBDVVP2 gene and ILTVgB, gD and gI genes as well as a construction method and application of the recombinant herpesvirus turkey strain, and belongs to the technical field of medicine or veterinary medicine. The recombinant herpesvirus of turkeys is obtained by co-transfecting CEF cells with recombinant clay containing H9 subtype AIVHA genes and ILTVgB genes, recombinant clay containing IBDVVP2 genes, recombinant clay containing ILTVgD and gI genes and parent clay containing HVT genome DNA fragments, and then carrying out virus rescue. The recombinant herpesvirus of turkeys is obtained by the following steps: cotransfecting CEF cells with recombinant clay containing H9 subtype AIVHA genes and ILTVgB genes, recombinant clay containing IBDVVP2 genes, recombinant clay containing ILTVgD and gI genes, and parent clay containing HVT genome DNA fragments. The recombinant virus strain obtained by the invention has good in-vitro replication ability and genetic stability. After the SPF chicken is immunized, the SPF chicken can obtain strong immune protection on H9 subtype AIV (avian influenza virus), IBDV (infectious bursal disease virus) and ILTV (Illuminal Low Temperature Virus). The invention provides a new technical means for simultaneously preventing the H9 subtype avian influenza, the infectious bursal disease and the infectious laryngotracheitis.

Description

Technical Field

[0001] The present invention relates to a recombinant turkey herpes virus which co-expresses H9 subtype avian influenza virus HA gene, chicken infectious bursal disease virus VP2 gene and chicken infectious laryngotracheitis virus gB, gD and gI genes and an application thereof in preparing a vaccine for simultaneously preventing H9 subtype avian influenza, chicken infectious bursal disease and chicken infectious laryngotracheitis, belonging 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 and 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. 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 controlled by inactivated vaccines. However, the isolation rate of the H9 subtype AIV in my country has increased year by year in recent years, indicating that traditional inactivated vaccine immunity 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 bursal disease (IBD) is an acute, highly contagious, immunosuppressive infectious disease caused by infectious bursal disease virus (IBDV) that mainly harms chicks. The IBDV genome consists of two double-stranded RNA segments. The VP2 protein encoded by segment A is the only capsid protein of IBDV, and is also the main virulence gene and host protective antigen of IBDV. The virus mainly invades the bursa of Fabricius, the immune organ of chickens, causing immune dysfunction in infected chickens and reducing the immune effect of vaccines. Clinical manifestations are depression, anorexia, diarrhea and severe weakness in large groups of chickens. The autopsy is characterized by atrophy, hemorrhage and yellow jelly-like infiltration of the bursa of Fabricius. In 1957, IBD first broke out in Gambro, Delaware, USA. The pathogen that caused the disease was later called IBDV classic strong virus. In 1982, my country first reported the prevalence of IBDV classic strong virus in Beijing, and then this type of strain was widely prevalent in most poultry-raising areas in my country.

[0004] Infectious laryngotracheitis (ILT) is an acute upper respiratory tract infectious disease of chickens caused by infectious laryngotracheitis virus (ILTV). It is common during the transition between spring and autumn, especially in adult chickens. ILTV infects the respiratory system of chickens and mainly replicates in the tracheal epithelium of chickens. Infected chickens show symptoms such as dyspnea, conjunctivitis, and hemoptysis when they become ill. The mortality rate of some virulent infections is as high as 100%. The disease was first reported in the United States in 1925 and is now distributed in major chicken-raising areas in the world. The disease spreads quickly and has a high mortality rate. It occurs and spreads in many areas of my country, causing great losses to my country's poultry industry. Studies on chickens immunized with ILTV vaccines have found that the body's cellular immunity plays a major role in resisting ILTV infection. Since inactivated vaccines cannot effectively induce cellular immunity and the immune effect is poor, at present, attenuated live vaccines are mainly used to control the disease at home and abroad. However, live attenuated vaccines have the risk of reversion to virulence and virus shedding, and because the ILTV vaccine strain can remain latent in the body of immunized chickens for a long time, it also increases the possibility of recombination between the vaccine virus and other strains, thereby generating new highly virulent strains. Therefore, the development of a safe, efficient, and risk-free new genetic engineering vaccine is of great significance for the prevention and control of ILT.

[0005] Herpesvirus of turkey (HVT) is a herpesvirus that is non-pathogenic to chickens and is widely used for 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 body 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

[0006] One of the purposes of the present invention is to provide a recombinant turkey herpes virus that co-expresses the HA gene of H9 subtype avian influenza virus, the VP2 gene of infectious bursal disease virus of chickens, and the gB, gD and gI genes of infectious laryngotracheitis virus of chickens;

[0007] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant turkey herpes virus;

[0008] The third object of the present invention is to provide the use of the above-mentioned recombinant turkey herpes virus in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bursal disease and infectious laryngotracheitis.

[0009] In order to achieve the above object, the present invention adopts the following technical means:

[0010] On the one hand, the present invention provides a recombinant turkey herpesvirus strain that co-expresses the H9 subtype avian influenza virus HA gene, the infectious bursal virus VP2 gene, and the infectious laryngotracheitis virus gB, gD, and gI genes. The recombinant turkey herpesvirus strain is obtained by inserting the expression framework CAGW-HAGB containing the H9 subtype AIV HA gene and the ILTV gB gene, the expression framework CMV-IBDVC2 containing the IBDV VP2 gene, and the expression framework ILTV-GDGI containing the ILTV gD and gI genes into the turkey herpesvirus strain genome.

[0011] In a preferred embodiment of the present invention, the turkey herpesvirus strain is HVT FC126 strain, and the GenBank accession number of its genomic DNA sequence is AF291866.

[0012] In a preferred embodiment of the present invention, the expression framework CAGW-HAGB comprising the H9 subtype AIV HA gene and the ILTV gB gene is inserted between nucleotides 95322-95323 of the HVT FC126 strain genome; the expression framework CMV-IBDVC2 comprising the IBDV VP2 gene is inserted between nucleotides 112071-112088 of the HVT FC126 strain genome; the expression framework ILTV-GDGI comprising the ILTV gD and gI genes is inserted between nucleotides 140079-140730 of the HVT FC126 strain genome.

[0013] In a preferred embodiment of the present invention, the expression framework CAGW-HAGB containing the H9 subtype AIV HA gene and the ILTV gB gene sequentially comprises a chicken β-actin promoter, an HA gene coding sequence, a porcine teschovirus 2A coding sequence, a gB gene coding sequence, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylic acid sequence, and its nucleotide sequence is shown in SEQ ID NO.4.

[0014] In a preferred embodiment of the present invention, the expression framework CMV-IBDVC2 comprises a mouse cytomegalovirus promoter, an IBDV VP2 gene coding sequence and an SV40 polyadenylation sequence, and its nucleotide sequence is shown in SEQ ID NO.6.

[0015] In a preferred embodiment of the present invention, the nucleotide sequence of the expression framework ILTV-GDGI is shown as SEQ ID NO.7.

[0016] On the other hand, the present invention also provides a method for constructing the recombinant turkey herpes virus strain, comprising the following steps:

[0017] (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0018] The genomic DNA of HVT FC126 strain was extracted and cloned into the pCC1Fos vector in segments. Six recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned the genomic DNA fragments of HVT FC126 strain and could be assembled to cover the complete HVT FC126 genome were selected; among them, H434 contained the nucleotide fragment of HVT FC126 genome at positions 1-40394, H481 contained the nucleotide fragment of HVT FC126 genome at positions 31586-69191, H159 contained the nucleotide fragment of HVT FC126 genome at positions 58293-92645, H483 contained the nucleotide fragment of HVT FC126 genome at positions 76940-110712, and H361 contained the nucleotide fragment of HVT The nucleotide fragment of FC126 genome is 103006-134393, and H220 contains the nucleotide fragment of HVT FC126 genome is 123621-159160;

[0019] (2) Construction of recombinant cosmids expressing H9 subtype AIV HA gene and ILTV gB gene

[0020] A recombinant plasmid comprising a chicken β-actin promoter, AIV HA gene and ILTV gB gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence expression frame CAGW-HAGB was constructed, wherein the HA gene and gB gene coding regions were connected by a porcine Teschovirus 2A self-cleavage peptide coding sequence; the expression frame CAGW-HAGB was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid H483 by using the Red / ET recombination method, and a recombinant cosmid that co-expressed the HA gene and the gB gene was constructed and named H483-53-HAGB;

[0021] (3) Construction of recombinant cosmid expressing IBDV VP2 gene

[0022] A recombinant expression plasmid comprising a mouse cytomegalovirus promoter, an IBDV VP2 gene coding sequence and an SV40 polyadenylation sequence expression frame CMV-IBDVC2 was constructed; the expression frame CMV-IBDVC2 was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid H361 by using the Red / ET recombination method to construct a recombinant cosmid expressing the IBDV VP2 gene, which was named H361-65-IBDVC2;

[0023] (4) Construction of recombinant cosmids expressing ILTV gD and gI genes

[0024] According to the genome sequence of ILTVWG strain with GenBank accession number JX458823, ILTV gD and gI gene expression framework ILTV-GDGI was obtained by PCR amplification, and the expression framework ILTV-GDGI was cloned into the recombinant cosmid H220 between nucleotides 140079 and 140730 of the HVT FC126 genome using the Red / ET recombination method to construct a recombinant cosmid expressing ILTV gD and gI genes, named H220-88-GDGI;

[0025] (5) Rescue of recombinant HVT expressing H9 subtype AIV HA gene, IBDV VP2 gene, and ILTV gB, gD, and gI genes

[0026] Recombinant cosmids H483-53-HAGB, H361-65-IBDVC2, H220-88-GDGI and parental cosmids H434, H481, and H159 cloned with HVTFC126 strain genomic fragments were extracted; the above-mentioned recombinant cosmids and parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method, and the viruses were harvested after plaque lesions appeared in culture. The recombinant HVT with H9 subtype AIV HA gene, IBDV VP2 gene, and ILTV gB, gD, and gI genes inserted into the HVTFC126 strain genome was rescued and named H30102.

[0027] In a preferred embodiment of the present invention, the nucleotide sequence of the expression framework CAGW-HAGB is shown as SEQ ID NO.4.

[0028] In a preferred embodiment of the present invention, the expression framework CMV-IBDVC2 comprises a mouse cytomegalovirus promoter, an IBDV VP2 gene coding sequence and an SV40 polyadenylation sequence, and its nucleotide sequence is shown in SEQ ID NO.6.

[0029] In a preferred embodiment of the present invention, the nucleotide sequence of the expression framework ILTV-GDGI is shown as SEQ ID NO.7.

[0030] On the other hand, the present invention also proposes the use of the recombinant turkey herpesvirus strain in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bursal disease and infectious laryngotracheitis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a recombinant turkey herpes virus that co-expresses the HA gene of H9 subtype avian influenza virus, the VP2 gene of infectious bursal disease virus of chickens, and the gB, gD and gI genes of infectious laryngotracheitis virus of chickens, and a construction method thereof, as well as the use of the recombinant turkey herpes virus strain in the preparation of a vaccine for preventing H9 subtype avian influenza, infectious bursal disease of chickens, and infectious laryngotracheitis of chickens. The recombinant turkey herpes virus of the present invention is obtained by co-transfecting CEF cells with a recombinant cosmid containing the HA gene of H9 subtype AIV and the gB gene of ILTV, a recombinant cosmid containing the VP2 gene of IBDV, a recombinant cosmid containing the gD and gI genes of ILTV, and a parent cosmid containing the genomic DNA fragment of HVT FC126, and then rescuing the virus. 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 the strong toxins of H9 subtype AIV, IBDV and ILTV. The invention provides a new technical means for preventing H9 subtype avian influenza, infectious bursal disease and infectious laryngotracheitis at the same time, and can achieve the effect of preventing multiple diseases with one injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The PCR identification results of the recombinant cosmids H483-53-HAGB, H361-65-IBDVC2, and H220-88-GDGI;

[0034] Figure 2 This is the plaque lesion produced by the recombinant virus H30102 on CEF;

[0035] Figure 3 This is the PCR identification result of the recombinant virus H30102 genomic DNA;

[0036] Figure 4 The results of the expression test of HA, VP2, gB, gD, and gI proteins in CEF infected with the recombinant virus H30102 are shown;

[0037] Figure 5 is the replication kinetic curve of the recombinant virus H30102 on CEF cells;

[0038] Figure 6 This is the PCR test result of the genetic stability of the recombinant virus H30102;

[0039] Figure 7 These are the results of indirect immunofluorescence tests of the 20th generation H30102 expressing HA, VP2, gB, gD, and gI proteins. DETAILED DESCRIPTION

[0040] 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.

[0041] Example 1: Construction and identification of recombinant HVT expressing H9 subtype AIV HA gene, IBDV VP2 gene and ILTV gB, gD, gI genes

[0042] 1.1 Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0043] Extract HVT FC126 strain genomic DNA, the GenBank accession number of the HVT FC126 strain genomic DNA sequence is AF291866. According to the instructions of the CopyControl Fosmid Library Production Kit, the FC126 strain genomic DNA was cloned into the pCC1Fos vector. The constructed recombinant cosmid was terminally sequenced using primers pCC1F and pCC1R (see Table 1 for details). According to the sequencing results, 6 recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned FC126 strain genomic DNA fragments and could be spliced ​​to cover the complete HVT FC126 genome were selected. Among them, H434 comprises the nucleotide fragment of 1-40394 position of FC126 genome, H481 comprises the nucleotide fragment of 31586-69191 position of FC126 genome, H159 comprises the nucleotide fragment of 58293-92645 position of FC126 genome, H483 comprises the nucleotide fragment of 76940-110712 position of FC126 genome, H361 comprises the nucleotide fragment of 103006-134393 position of FC126 genome, and H220 comprises the nucleotide fragment of 123621-159160 position of FC126 genome. The above-mentioned 6 recombinant clays cloned with FC126 genomic DNA fragments are extracted, and 6 recombinant clays are co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. The appearance of cytopathic effect can be observed after transfection for 4-5 days, that is, the parental virus strain HVT FC126 is rescued.

[0044] 1.2 Construction and identification of recombinant cosmids expressing H9 AIV HA gene and ILTV gB gene

[0045] According to the HA gene coding region sequence of the H9 subtype AIV TJ strain isolated and identified in this laboratory, the porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence was added to its 3' end, and the target gene H9HA (SEQ ID NO.1) was synthesized after chicken codon optimization. According to the ILTV WG strain genome sequence (GenBank accession number JX458823), the ILTV gB gene (SEQ ID NO.2) was synthesized after chicken codon optimization design. The target gene fragment HAGB (shown in SEQ ID NO.3) containing the HA and gB gene coding sequences was obtained by fusion PCR amplification using primers H9P1F, H9LTP1R, H9LTP2F and LTP2R (Table 1), wherein the HA gene and gB gene coding regions are connected by the self-cleavage peptide P2A coding sequence. The PCR product of the target gene HAGB was purified and cloned into the downstream of the chicken β-actin promoter of the pCAGGS vector. At the same time, the woodchuck hepatitis virus post-transcriptional regulatory sequence (GenBank accession number MQ208857.1) was inserted between the target gene HAGB and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-HAGB for co-expressing the H9AIV HA gene and the ILTV gB gene, wherein the HAGB gene expression framework, named CAGW-HAGB, sequentially comprises: chicken β-actin promoter, HA gene coding sequence, P2A coding sequence, gB gene coding sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit β-globulin polyadenylation sequence, and its nucleotide sequence is shown in SEQ ID NO. 4. The recombinant plasmid was sequenced using primers CAGF and CAGR (Table 1), and the nucleotide sequence of the target gene HAGB obtained was shown in SEQ ID NO. 3, which was consistent with expectations, indicating that the recombinant plasmid was constructed correctly.

[0046] According to the instructions of the Counter Selection BAC Modification Kit, the HAGB gene expression framework CAGW-HAGB (shown in SEQ ID NO.4) was cloned into the 95322-95323 nucleotides of the HVT FC126 genome in the recombinant cosmid H483 using the Red / ET recombination method to construct the recombinant cosmid H483-53-HAGB that co-expresses the HA gene and gB gene. The target gene-specific primer LT1348F and the target gene downstream homology arm primer HVT54R (Table 1) were used to identify whether the recombinant cosmid H483-53-HAGB was inserted into the target gene expression framework by PCR. The results showed that the PCR fragment of about 2687 bp was obtained ( Figure 1), which was consistent with expectations; sequencing results showed that the PCR product contained the gB gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The parent cosmid H483 had no target gene insertion sequence, and the PCR result was negative. The above results showed that the recombinant cosmid H483-53-HAGB was constructed correctly.

[0047] 1.3 Construction and identification of recombinant cosmids expressing IBDV VP2 gene

[0048] According to the VP2 gene sequence of IBDVFaragher 52 / 70 strain (GenBank accession number HG974565), the target gene IBDVC2 was synthesized, and its nucleotide sequence is shown in SEQ ID NO.5. The recombinant plasmid pUC57-IBDVC2 cloned with the IBDVC2 gene was used as a template, and the IBDVC2 gene was obtained by PCR amplification using primers IBDVC2F and IBDVC2R. The purified PCR product was cloned into the downstream of the mouse cytomegalovirus promoter of the pCMV vector to construct a recombinant plasmid pCMV-IBDVC2 expressing the IBDV VP2 gene.

[0049] According to the instructions of the Counter Selection BAC Modification Kit, the expression framework CMV-IBDVC2 (shown in SEQ ID NO.6) containing the mouse cytomegalovirus promoter, the IBDV VP2 gene coding sequence and the SV40 polyadenylation sequence was cloned into the recombinant cosmid H361 between nucleotides 112071-112088 of the HVT FC126 genome using the Red / ET recombination method to construct the recombinant cosmid H361-65-IBDVC2 expressing the IBDV VP2 gene. The target gene primer IBDVC2F and the target gene downstream homology arm primer HVT65F were used to identify whether the recombinant cosmid H361-65-IBDVC2 was inserted into the target gene expression framework, and the PCR product was sequenced and identified. The results showed that a PCR fragment of about 1800 bp was obtained ( Figure 1 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the IBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The above results showed that the recombinant cosmid H361-65-IBDVC2 was constructed correctly.

[0050] 1.4 Construction and identification of recombinant cosmids expressing ILTV gD and gI genes

[0051] According to the genome sequence of ILTVWG strain (GenBank accession number JX458823), ILTV gD and gI gene expression framework ILTV-GDGI was amplified by PCR, and its nucleotide sequence is shown in SEQ ID NO.7. According to the instructions of Counter Selection BACModification Kit, the expression framework ILTV-GDGI was used to replace the nucleotides 140080-140729 of the HVT FC126 genome in the recombinant cosmid H220 by the Red / ET recombination method to construct the recombinant cosmid H220-88-GDGI expressing ILTV gD and gI genes. The target gene primer ILTVgIF and the target gene downstream homology arm primer HVT88R were used to identify whether the target gene expression framework was inserted into the recombinant cosmid H220-88-GDGI by PCR, and the PCR product was sequenced and identified. The results showed that the PCR fragment with a size of about 1700bp ( Figure 1 ), which was consistent with expectations. The recombinant cosmid was amplified by PCR and sequenced using primers HVT88F and HVT88R. The sequencing results showed that the ILTV gD and gI genes were correctly inserted into the HVT FC126 genome, and the target gene sequence was correct. The above results indicate that the recombinant cosmid H220-88-GDGI was constructed correctly.

[0052] Table 1 PCR primers used to construct and identify recombinant cosmids

[0053]

[0054]

[0055] 1.5 Rescue and identification of recombinant HVT expressing H9 subtype AIV HA gene, IBDV VP2 gene, and ILTV gB, gD, and gI genes

[0056] The recombinant cosmids H483-53-HAGB, H361-65-IBDVC2, H220-88-GDGI and other parental cosmids H434, H481, H159 cloned with HVT FC126 genome fragments were extracted using a plasmid extraction kit. The above recombinant cosmids and parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method. The virus was harvested after 4-5 days of culture and plaque lesions appeared. The virus was continuously passaged and preserved in CEF cells to rescue the recombinant virus in which the H9 subtype AIV HA gene, IBDV VP2 gene, and ILTVgB, gD, and gI genes were simultaneously inserted into the HVT FC126 genome. The virus was named H30102. The plaque lesions produced by the above recombinant virus on CEF are shown in Figure 2. Figure 2 shown.

[0057] The genomic DNA of the recombinant virus H30102 was extracted, and the genomic DNA of the parent virus HVTFC126 was used as a control. The recombinant virus was identified by PCR using the target gene HAGB specific primer LT1348F and the downstream homology arm primer HVT54R (Table 1). The results showed that the recombinant virus H30102 amplified a fragment of about 2687 bp, which was consistent with expectations ( Figure 3 ). The recombinant virus was identified by PCR using the IBDVVP2 gene specific primer IBDVC2F and the downstream homology arm primer HVT65F. The results showed that the recombinant virus H30102 amplified a PCR fragment of about 1800 bp ( Figure 1 ), which was consistent with expectations. The recombinant viral genomic DNA was identified by PCR using the ILTV gI specific primer ILTVgIF and the downstream homology arm primer HVT88R. The result was amplification of a PCR fragment of about 1700 bp ( Figure 3 ), which was consistent with expectations. The parent virus HVT FC126 had no target gene insertion, and the PCR result was negative. The above results showed that the target genes HAGB, IBDVC2 and GDGI were correctly inserted into the genome of the HVT FC126 strain, and the recombinant virus H30102 was correctly constructed.

[0058] Example 2: In vitro biological characteristics analysis of recombinant turkey herpesvirus expressing H9 subtype AIV HA gene, IBDV VP2 gene and ILTV gB, gD, gI genes

[0059] 2.1 Detection of recombinant viruses expressing H9 subtype AIV HA, IBDV VP2, and ILTV gB, gD, and gI proteins

[0060] The recombinant virus H30102 and the parental virus HVT FC126 strain were inoculated into CEF cells cultured in 6-well plates, with each virus strain inoculated into 5 wells, and non-infected cells were set as controls. After 3-4 days of culture and the appearance of plaque lesions, the cells were fixed with anhydrous ethanol, and the expression of HA, VP2, gB, gD, gI polyclonal antibodies and VP2 monoclonal antibodies were used as primary antibodies, and FITC-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies. Indirect immunofluorescence assay was used to detect the expression of HA, VP2, gB, gD, and gI proteins, and CEF infected with the parental virus HVT FC126 was used as a negative control. The results showed that cells infected with the recombinant virus H30102 could react with the HA, VP2, gB, gD, and gI detection antibodies, showing green fluorescence signals ( Figure 4). No fluorescence was observed in cells infected with the parent virus HVT FC126 strain and in control cells that were not infected. The above results indicate that the recombinant virus H30102 can co-express HA, VP2, gB, gD, and gI proteins in infected cells.

[0061] 2.2 Analysis of in vitro replication characteristics of recombinant virus H30102

[0062] The recombinant virus H30102 and the parent virus HVTFC126 were inoculated on CEF in a 6-well plate at a dose of 100 plaque-forming units (PFU). After infection, the virus-containing cells were collected every 24 hours 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 was drawn to analyze the in vitro replication characteristics of the recombinant virus H30102 and the parent virus HVT FC126 in CEF. The results showed that the replication titers of the recombinant virus H30102 and the parent virus HVT FC126 reached the highest peak at 120 hours after infection, which were 1.86×10 5 PFU / ml and 1.95×10 5 PFU / ml, the titer of the recombinant virus at each time point after infection of CEF was not significantly different from that of the parental virus (P>0.05)( Figure 5 ). The above results show that the recombinant virus H30102 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus HVT FC126 strain.

[0063] 2.3 Genetic stability test of recombinant virus H30102

[0064] The rescued recombinant virus H30102 was continuously passaged on CEF to the 20th generation, and the genomic DNA of the 20th generation recombinant virus was extracted for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the recombinant virus genome. The results showed that the 20th generation recombinant virus was identified by PCR using the target gene HAGB specific primer LT1348F and the downstream homology arm primer HVT54R (Table 1), and a fragment of about 2687 bp could be amplified, which was consistent with expectations ( Figure 6 ); The recombinant virus genomic DNA was amplified and sequenced using primers CAGF and CAGR, and the sequence of the target gene HAGB inserted into the HVTFC126 genome was found to be correct. The 20th generation recombinant virus was identified by PCR using the VP2 gene primer IBDVC2F and the downstream homology arm primer HVT65F, and a PCR fragment of about 1800 bp was amplified ( Figure 1), which was consistent with expectations; sequencing results showed that the PCR product contained the IBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The 20th generation recombinant virus genomic DNA was identified by PCR using the target gene GDGI specific primer ILTVgIF and the downstream homology arm primer HVT88R. The result was amplified to obtain a PCR fragment of about 1700 bp ( Figure 6 ), the size was correct; the recombinant viral genomic DNA was amplified by PCR and sequenced using primers HVT88F and HVT88R (Table 1), and the results showed that the ILTV gD and gI gene expression framework sequences inserted into the HVT FC126 genome were correct.

[0065] At the same time, the 20th generation recombinant virus was inoculated into CEF cells, and indirect immunofluorescence tests were performed using HA, VP2, gB, gD, and gI specific antibodies to detect the stability of target gene expression. The results showed that after the recombinant virus H30102 was continuously propagated on CEF cells to the 20th generation, the exogenous target genes HA, VP2, gB, gD, and gI could still be stably expressed ( Figure 7 ). The above results show that the HA, VP2, gB, gD, and gI genes inserted into the HVT FC126 genome can stably exist during the virus passage process, and the recombinant virus H30102 has good genetic stability.

[0066] Example 3: Safety and immunogenicity testing of recombinant virus H30102

[0067] 3.1 Safety testing of recombinant virus H30102

[0068] The fifth-generation recombinant virus H30102 was inoculated into one-day-old SPF chickens at a dose of 4000 PFU / chicken, and the clinical symptoms of the chickens in each group were observed every day after inoculation. 28 days after inoculation, 5 test chickens were randomly selected from each group, and their weights were weighed to evaluate the effect of the recombinant virus H30102 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the organs such as the bursa of Fabricius, thymus, spleen, and liver were collected, weighed, and observed for atrophy or swelling. The results showed that the recombinant virus H30102 did not cause adverse clinical reactions in the test chickens after inoculation of SPF chickens, and the feeding and drinking water of the immunized chickens were normal. 28 days after inoculation, the weighing and autopsy results showed that the weight of the chickens in the recombinant virus H30102 inoculation group was no significantly different from that of the normal uninoculated control group; the test chickens were autopsied, 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 virus H30102 is safe for SPF chickens.

[0069] 3.2 Immunoprotective test of recombinant virus H30102 against H9 subtype AIV

[0070] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 chicks in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30102 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parent virus HVT FC126 at the same dose, and Group 3 was not immunized as a blank control group. 28 days after immunization, Groups 1-2 were nasally challenged with the H9 subtype AIV LC18 strain; Group 3 was not challenged and served as a healthy control group. After the challenge, the chickens were observed for 14 days and the clinical symptoms were recorded. Five days after the challenge, the laryngeal and cloacal swabs of each chicken in each group were collected for virus isolation. The collected cotton swabs were repeatedly frozen and thawed three times and then centrifuged. The supernatant was taken and 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 the allantoic fluid was collected to determine the HA titer. The titer was not less than 1:16 and was judged as positive for virus isolation; the samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0071] The results showed that after the experimental chickens were challenged with H9 subtype AIV, the virus isolation of 10 chickens in the parental virus HVT FC126 strain inoculation group was positive, and the virus isolation of the experimental chickens in the blank control group without challenge was negative, and the challenge test was established. After the 10 chickens in the recombinant virus H30102 inoculation group were challenged with H9 subtype AIV, during the observation period, all chickens' feeding, drinking, mental state, movement, etc. were normal, with no obvious adverse clinical symptoms, and virus isolation was negative. The above results show that the protection rate of the recombinant virus H30102 against H9 subtype AIV is 100%.

[0072] 3.3 Immunoprotective test of recombinant virus H30102 against ILTV virulent virus

[0073] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30102 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parental virus HVT FC126 at the same dose, and Group 3 was not immunized as a blank control group. 28 days after immunization, Groups 1-2 were challenged with the virulent WG strain of ILTV respectively; 14 days after the challenge, the clinical symptoms of the test chickens were recorded, and the survival of the chickens in each group was counted; 14 days after the challenge, the test chickens were autopsied to detect the lesions of the larynx, trachea and organs of the test chickens. The results showed that after the parental virus HVT FC126 strain inoculation group was challenged with the virulent ILTV, all 10 test chickens showed symptoms such as lethargy, messy feathers, conjunctivitis, head shaking, tearing, and dyspnea, of which 6 died; the autopsy showed that the diseased chickens had laryngeal and tracheal bleeding, and had lesions such as cheesy exudates. After the 10 chickens in the recombinant virus H30102 inoculation group were challenged with ILTV strong virus, one chicken showed symptoms of depression and dyspnea, and laryngeal hemorrhagic lesions were found in the autopsy, while the other 9 chickens did not show adverse clinical symptoms and autopsy lesions. The above results show that the protection rate of the recombinant virus H30102 against ILTV strong virus is 90%.

[0074] 3.4 Immunoprotective test of recombinant virus H30102 against IBDV super-virulent virus

[0075] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30102 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parent virus HVT FC126 at the same dose, and Group 3 was not inoculated as a blank control group. 28 days after immunization, Group 1 and Group 2 were challenged with the super-virulent HN strain of IBDV, respectively, and observed for 7 days after the challenge, and the clinical symptoms of the test chickens were recorded, and the survival was counted; 7 days after the challenge, the bursa of Fabricius was observed for atrophy, hemorrhage, or yellow jelly-like infiltration. The results showed that after the parental virus HVT FC126 inoculation group was challenged with IBDV super-virulent virus, all 10 chickens showed symptoms such as listlessness, disheveled feathers, and lying on the ground. Eight of the infected chickens died, and autopsy showed that the bursa of Fabricius of all 10 chickens was atrophic, with strip-like bleeding or yellow jelly-like infiltration lesions; all 10 chickens in the recombinant virus H30102 inoculation group survived after being challenged with IBDV super-virulent virus, and autopsy found no obvious lesions in the bursa of Fabricius. The above results show that the protection rate of the recombinant virus H30102 against IBDV super-virulent virus is 100%.

Claims

1. A recombinant turkey herpesvirus strain (HVT) co-expressing the HA gene of H9 subtype avian influenza virus (AIV), the VP2 gene of infectious bursal disease virus (IBDV) and the gB, gD and gI genes of infectious laryngotracheitis virus (ILTV), characterized in that: The recombinant turkey herpesvirus strain is obtained by inserting the expression frame CAGW-HAGB containing H9 subtype AIV HA gene and ILTV gB gene, the expression frame CMV-IBDVC2 containing IBDVVP2 gene and the expression frame ILTV-GDGI containing ILTVgD and gI genes into the turkey herpesvirus strain genome.

2. The recombinant turkey herpes virus strain according to claim 1, characterized in that The turkey herpes virus strain is HVT FC126 strain, and the GenBank accession number of its genomic DNA sequence is AF291866.

3. The recombinant turkey herpes virus strain according to claim 1, characterized in that The expression frame CAGW-HAGB containing the H9 subtype AIVHA gene and the ILTV gB gene is inserted between the 95322-95323 nucleotides of the HVT FC126 strain genome; the expression frame CMV-IBDVC2 containing the IBDVVP2 gene is inserted between the 112071-112088 nucleotides of the HVTFC126 strain genome; the expression frame ILTV-GDGI containing the ILTV gD and gI genes is inserted between the 140079-140730 nucleotides of the HVT FC126 strain genome.

4. The recombinant turkey herpes virus strain according to claim 3, characterized in that The expression framework CAGW-HAGB containing the H9 subtype AIVHA gene and the ILTV gB gene sequentially contains a chicken β-actin promoter, a HA gene coding sequence, a porcine teschovirus 2A coding sequence, a gB gene coding sequence, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylic acid sequence, and its nucleotide sequence is shown in SEQ ID NO.

4.

5. The recombinant turkey herpes virus strain according to claim 3, characterized in that The expression framework CMV-IBDVC2 comprises a mouse cytomegalovirus promoter, an IBDVVP2 gene coding sequence and an SV40 polyadenylation sequence, and its nucleotide sequence is shown in SEQ ID NO.

6.

6. The recombinant turkey herpes virus strain according to claim 3, characterized in that The nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.

7.

7. A method for constructing the recombinant turkey herpes virus strain according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain The genomic DNA of HVT FC126 strain was extracted and cloned into the pCC1Fos vector in segments. Six recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned the genomic DNA fragments of HVT FC126 strain and could be assembled to cover the complete HVT FC126 genome were selected; among them, H434 contained the nucleotide fragment of HVT FC126 genome at positions 1-40394, H481 contained the nucleotide fragment of HVT FC126 genome at positions 31586-69191, H159 contained the nucleotide fragment of HVT FC126 genome at positions 58293-92645, H483 contained the nucleotide fragment of HVT FC126 genome at positions 76940-110712, and H361 contained the nucleotide fragment of HVT The nucleotide fragment of FC126 genome is 103006-134393, and H220 contains the nucleotide fragment of HVT FC126 genome is 123621-159160; (2) Construction of recombinant cosmids expressing H9 subtype AIVHA gene and ILTV gB gene A recombinant plasmid comprising a chicken β-actin promoter, AIVHA gene and ILTV gB gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence expression frame CAGW-HAGB is constructed, wherein the HA gene and gB gene coding regions are connected by a porcine Teschovirus 2A self-cleavage peptide coding sequence; the expression frame CAGW-HAGB is cloned into the 95322-95323 nucleotides of the HVT FC126 strain genome in the recombinant cosmid H483 by using the Red / ET recombination method, and a recombinant cosmid that co-expresses the HA gene and the gB gene is constructed and named H483-53-HAGB; (3) Construction of recombinant cosmid expressing IBDV VP2 gene A recombinant expression plasmid comprising a mouse cytomegalovirus promoter, an IBDVVP2 gene coding sequence and an SV40 polyadenylation sequence expression frame CMV-IBDVC2 was constructed; the expression frame CMV-IBDVC2 was cloned into the recombinant cosmid H361 between nucleotides 112071 and 112088 of the HVT FC126 strain genome using the Red / ET recombination method to construct a recombinant cosmid expressing the IBDVVP2 gene, named H361-65-IBDVC2; (4) Construction of recombinant cosmids expressing ILTV gD and gI genes According to the genome sequence of ILTVWG strain with GenBank accession number JX458823, ILTV gD and gI gene expression framework ILTV-GDGI was obtained by PCR amplification, and the expression framework ILTV-GDGI was cloned into the recombinant cosmid H220 between nucleotides 140079 and 140730 of the HVT FC126 strain genome by Red / ET recombination method to construct a recombinant cosmid expressing ILTV gD and gI genes, named H220-88-GDGI; (5) Rescue of recombinant HVT expressing H9 subtype AIVHA gene, IBDVVP2 gene, and ILTVgB, gD, and gI genes Recombinant cosmids H483-53-HAGB, H361-65-IBDVC2, H220-88-GDGI and parental cosmids H434, H481, and H159 cloned with HVT FC126 strain genomic fragments were extracted; the above-mentioned recombinant cosmids and parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method, and the viruses were harvested after plaque lesions appeared in culture. The recombinant HVT with H9 subtype AIV HA gene, IBDVVP2 gene, and ILTV gB, gD, and gI genes inserted into the HVT FC126 strain genome was rescued and named H30102.

8. The method according to claim 7, characterized in that The nucleotide sequence of the expression framework CAGW-HAGB is shown in SEQ ID NO.4; the nucleotide sequence of the expression framework CMV-IBDVC2 is shown in SEQ ID NO.6; and the nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.

7.

9. Use of the recombinant turkey herpesvirus strain according to any one of claims 1 to 6 in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bursal disease and infectious laryngotracheitis.

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