Recombinant turkey herpesvirus strain as well as construction method and application thereof

Abstract

The invention discloses a recombinant turkey herpesvirus strain as well as a construction method and application thereof, and belongs to the technical field of medicine or veterinary medicine. The recombinant turkey herpesvirus is a recombinant turkey herpesvirus strain co-expressing a Newcastle disease virus F gene and infectious laryngotracheitis virus gB, gD and gI genes or a recombinant turkey herpesvirus strain co-expressing a Newcastle disease virus F gene, a chicken infectious bursal disease virus VP2 gene and infectious laryngotracheitis virus gB, gD and gI genes. The recombinant turkey herpesvirus strain obtained by the invention has good in-vitro replication ability and genetic stability. Safety and immunogenicity experiments prove that the two recombinant turkey herpesvirus strains are safe to SPF chickens and have good immune protection effects on NDV virulent strains, ILTV virulent strains and IBDV virulent strains. The invention provides an effective technical means for simultaneously preventing the Newcastle disease and the chicken infectious laryngotracheitis or simultaneously preventing the Newcastle disease, the chicken infectious bursal disease and the chicken infectious laryngotracheitis.

Description

Technical Field

[0001] The present invention relates to a recombinant turkey herpes virus co-expressing Newcastle disease virus F gene and infectious laryngotracheitis virus gB, gD and gI genes and its application in preparing Newcastle disease and infectious laryngotracheitis vaccines, and also relates to a recombinant turkey herpes virus co-expressing Newcastle disease virus F gene, infectious bursal disease virus VP2 gene and infectious laryngotracheitis virus gB, gD and gI genes and its application in preparing Newcastle disease, infectious bursal disease and infectious laryngotracheitis vaccines. The present invention belongs to the technical field of medicine or veterinary medicine. Background Art

[0002] Newcastle disease (ND) is a virulent, highly contact, respiratory infectious disease of poultry caused by Newcastle disease virus (NDV). At present, Newcastle disease is distributed globally, causing huge losses to the aquaculture industry and the import and export trade of related products around the world. The NDV currently prevalent in my country is mainly the genotype VII subtype. The genome of NDV is a non-segmented, single-stranded negative-strand RNA, in which the F protein is a glycoprotein on the NDV envelope, which is the main virulence gene and host protective antigen of NDV. At present, ND is mainly prevented and controlled by inactivated vaccines and attenuated live vaccines. Since the immune effect of attenuated live vaccines is easily interfered by maternal antibodies, and the immune duration of inactivated vaccines is not long, the local ND vaccine must be immunized multiple times to achieve the immune effect, thereby increasing the immune pressure and breeding burden of the chicken flock. Therefore, the current market urgently needs a new ND vaccine that is homologous to the currently popular genotype VII strain, is not interfered by maternal antibodies, is suitable for early vaccination of chicks, and has a long immune duration.

[0003] 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 after some strong toxins is as high as 100%. Autopsy shows swelling, congestion, and bleeding of the laryngeal and tracheal mucosa, sometimes with yellow cheesy substances. The disease was first reported in the United States in 1925 and is now distributed in major chicken-raising areas around 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 have poor immune effects, live attenuated vaccines are currently used to control the disease. However, live attenuated vaccines have the risk of reversion to virulence and shedding of the virus, and since 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 non-risk-of-reversion new genetic engineering vaccine is of great significance for the prevention and control of ILT.

[0004] 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.

[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 Newcastle disease virus F gene and the infectious laryngotracheitis virus gB, gD and gI genes, and a method for constructing the recombinant turkey herpes virus and its use in preparing Newcastle disease and infectious laryngotracheitis vaccines;

[0007] The second object of the present invention is to provide a recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene, the infectious bursal disease virus VP2 gene, and the infectious laryngotracheitis virus gB, gD and gI genes, and a construction method thereof, and an application thereof in the preparation of Newcastle disease, infectious bursal disease and infectious laryngotracheitis vaccines.

[0008] In order to achieve the above object, the present invention adopts the following technical means:

[0009] On the one hand, the present invention provides a recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene and the infectious laryngotracheitis virus gB, gD, and gI genes, which is obtained by inserting an expression frame CAGW-F2AGB containing the NDVF gene and the ILTVgB gene and an expression frame ILTV-GDGI containing the ILTV gD and gI genes into the turkey herpesvirus genome.

[0010] In a specific embodiment of the present invention, the turkey herpesvirus is HVT FC126 strain, and the GenBank accession number of the genomic DNA sequence of HVTFC126 strain is AF291866.

[0011] In a specific embodiment of the present invention, the expression frame CAGW-F2AGB is inserted between nucleotides 95322-95323 of the HVT FC126 genome; the expression frame ILTV-GDGI is inserted between nucleotides 140079-140730 of the HVT FC126 genome.

[0012] In a specific embodiment of the present invention, the expression frame CAGW-F2AGB comprises a chicken β-actin promoter, NDVF gene and ILTV gB gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylic acid sequence, wherein the NDVF gene and ILTVgB gene coding sequences are connected by a self-cleaving peptide P2A coding sequence. Preferably, the nucleotide sequence of the expression frame CAGW-F2AGB is shown in SEQ ID NO.4. Preferably, the nucleotide sequence of the expression frame ILTV-GDGI is shown in SEQ ID NO.5.

[0013] Furthermore, the present invention also provides a method for constructing the recombinant turkey herpes virus strain that co-expresses the Newcastle disease virus F gene and the infectious laryngotracheitis virus gB, gD, and gI genes, comprising the following steps:

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

[0015] The genomic DNA of HVT FC126 strain was extracted and cloned into the pCC1Fos vector in segments. Six recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05 and HVT06, which cloned the genomic DNA fragments of HVT FC126 strain and could be assembled to cover the complete genome of HVT FC126 strain, were selected; among them, HVT01 contained the nucleotide fragment of 1-30189 of the genome of HVT FC126 strain, HVT02 contained the nucleotide fragment of 21572-61476 of the genome of HVT FC126 strain, HVT03 contained the nucleotide fragment of 50871-90478 of the genome of HVT FC126 strain, HVT04 contained the nucleotide fragment of 73762-108139 of the genome of HVT FC126 strain, and HVT05 contained the nucleotide fragment of 50871-90478 of the genome of HVT FC126 strain. The nucleotide fragment of the FC126 strain genome is 96776-135815, and HVT06 contains the nucleotide fragment of the HVT FC126 strain genome is 129602-159160;

[0016] (2) Construction of recombinant cosmids expressing NDVF and ILTVgB genes

[0017] A recombinant plasmid comprising a chicken β-actin promoter, NDVF 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-F2AGB was constructed, wherein the F gene and gB gene coding regions were connected by a P2A self-cleaving peptide coding sequence; the expression frame CAGW-F2AGB was cloned into the 95322-95323 nucleotides of the HVT FC126 strain genome in the recombinant cosmid HVT04 by using the Red / ET recombination method, and a recombinant cosmid that co-expressed the F gene and the gB gene was constructed and named HVT04-53-F2AGB;

[0018] (3) Construction of recombinant cosmids expressing ILTV gD and gI genes

[0019] 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 HVT06 between nucleotides 140079-140730 of the HVT FC126 strain genome by Red / ET recombination method to construct a recombinant cosmid expressing ILTV gD and gI genes, named HVT06-88-GDGI;

[0020] (4) Rescue of recombinant HVT expressing NDVF gene and ILTVgB, gD, and gI genes

[0021] The recombinant cosmids HVT04-53-F2AGB, HVT06-88-GDGI and the parental cosmids HVT01, HVT02, HVT03 and HVT05 cloned with the genome fragment of HVT FC126 strain were extracted by using a plasmid extraction kit; the recombinant cosmids and the parental cosmids were co-transfected into CEF cells by using the calcium phosphate transfection method, and the virus was harvested after the plaque lesions appeared in the culture, and the recombinant turkey herpes virus strain with the expression frame CAGW-F2AGB inserted between the 95322-95323 nucleotides of the HVTFC126 strain genome and the expression frame ILTV-GDGI inserted between the 140079-140730 nucleotides of the HVTFC126 strain genome was rescued and named H20501. Further, the present invention also provides the use of the recombinant turkey herpes virus strain in the preparation of a vaccine for preventing Newcastle disease and infectious laryngotracheitis at the same time.

[0022] On the other hand, the present invention provides a recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene, the infectious bursal disease virus VP2 gene, and the infectious laryngotracheitis virus gB, gD, and gI genes. The recombinant turkey herpesvirus strain is obtained by further inserting an expression framework CMV-IBDVC2 containing the IBDVVP2 gene into the turkey herpesvirus genome on the basis of the above-mentioned recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene and the infectious laryngotracheitis virus gB, gD, and gI genes, that is, the recombinant turkey herpesvirus strain is obtained by inserting the expression framework CAGW-F2AGB containing the NDVF gene and the ILTV gB gene, the expression framework CMV-IBDVC2 containing the IBDVVP2 gene, and the expression framework ILTV-GDGI containing the ILTVgD and gI genes into the HVT genome.

[0023] In another specific embodiment of the present invention, the turkey herpesvirus is HVT FC126 strain, and the GenBank accession number of the genomic DNA sequence of HVT FC126 strain is AF291866.

[0024] In another specific embodiment of the present invention, the expression frame CAGW-F2AGB is inserted between nucleotides 95322-95323 of the HVTFC126 genome; the expression frame CMV-IBDVC2 is inserted between nucleotides 112071-112088 of the HVTFC126 genome; and the expression frame ILTV-GDGI is inserted between nucleotides 140079-140730 of the HVTFC126 genome.

[0025] In another specific embodiment of the present invention, the expression frame CAGW-F2AGB comprises a chicken β-actin promoter, NDVF gene and ILTVgB gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylic acid sequence, wherein the NDVF gene and ILTVgB gene coding sequences are connected by a self-cleaving peptide P2A coding sequence. Preferably, the nucleotide sequence of the expression frame CAGW-F2AGB is as shown in SEQ ID NO.4.

[0026] In another specific embodiment of the present invention, the expression framework ILTV-GDGI comprises ILTVgD and gI gene coding sequences, and its nucleotide sequence is shown in SEQ ID NO.5.

[0027] In another specific embodiment of the present invention, the expression framework CMV-IBDVC2 comprises a mouse cytomegalovirus promoter, an IBDV classic virulent strain VP2 gene and an SV40 polyadenylation sequence, and its nucleotide sequence is shown in SEQ ID NO.7.

[0028] Furthermore, the present invention also proposes a method for constructing the recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene, the infectious bursal disease virus VP2 gene, and the infectious laryngotracheitis virus gB, gD, and gI genes, characterized in that it comprises the following steps:

[0029] (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0030] 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 genome were selected; among them, H434 contained the nucleotide fragment of 1-40394 of the genome of HVT FC126 strain, H481 contained the nucleotide fragment of 31586-69191 of the genome of HVT FC126 strain, H159 contained the nucleotide fragment of 58293-92645 of the genome of HVT FC126 strain, H483 contained the nucleotide fragment of 76940-110712 of the genome of HVT FC126 strain, and H361 contained the nucleotide fragment of 58293-92645 of the genome of HVT FC126 strain. The nucleotide fragment of the FC126 strain genome is 103006-134393, and H220 contains the nucleotide fragment of the HVT FC126 strain genome at 123621-159160;

[0031] (2) Construction of recombinant cosmids expressing NDVF and ILTVgB genes

[0032] A recombinant plasmid comprising a chicken β-actin promoter, NDVF 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-F2AGB is constructed, wherein the F gene and gB gene coding regions are connected by a P2A self-cleaving peptide coding sequence; the expression frame CAGW-F2AGB 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 F gene and the gB gene is constructed and named H483-53-F2AGB;

[0033] (3) Construction of recombinant cosmid expressing IBDV VP2 gene

[0034] 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;

[0035] (4) Construction of recombinant cosmids expressing ILTV gD and gI genes

[0036] 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;

[0037] (5) Rescue of recombinant HVT expressing NDVF gene, IBDVVP2 gene, and ILTV gB, gD, and gI genes

[0038] The recombinant cosmids H483-53-F2AGB, H361-65-IBDVC2, H220-88-GDGI and the parental cosmids H434, H481, and H159 cloned with the HVTFC126 strain genome fragment 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, and the viruses were harvested after the appearance of plaque lesions in culture, and the recombinant HVT with the NDVF gene, IBDVVP2 gene, and ILTVgB, gD, and gI genes inserted into the HVTFC126 strain genome was rescued and named H30301.

[0039] Furthermore, the present invention also provides the use of the recombinant turkey herpesvirus strain in the preparation of a vaccine for simultaneously preventing Newcastle disease, infectious bursal disease and infectious laryngotracheitis.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides a recombinant turkey herpes virus strain that co-expresses Newcastle disease virus F gene and infectious laryngotracheitis virus gB, gD and gI genes, and a recombinant turkey herpes virus strain that co-expresses Newcastle disease virus F gene, infectious bursal disease virus VP2 gene and infectious laryngotracheitis virus gB, gD and gI genes, and also provides the use of the above two recombinant turkey herpes virus strains in preventing Newcastle disease and infectious laryngotracheitis at the same time and preventing Newcastle disease, infectious bursal disease and infectious laryngotracheitis at the same time. The two recombinant turkey herpes virus strains provided by the present invention both have good replication ability on CEF and have good genetic stability. The two recombinant turkey herpes virus strains are safe for SPF chickens and have good immune protection effects on NDV strong toxins, ILTV strong toxins and IBDV super strong strains. The invention provides an effective technical means for simultaneously preventing Newcastle disease and infectious laryngotracheitis or simultaneously preventing Newcastle disease, infectious bursal disease and infectious laryngotracheitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The PCR identification results of the recombinant cosmids HVT04-53-F2AGB and HVT06-88-GDGI;

[0043] Figure 2 The plaque lesions produced by the recombinant turkey herpesvirus strain H20501 on CEF;

[0044] Figure 3 This is the result of PCR identification of the genomic DNA of the recombinant turkey herpesvirus strain H20501;

[0045] Figure 4 The results of the expression test of F, gB, gD, and gI proteins in CEF infected with the recombinant turkey herpesvirus strain H20501;

[0046] Figure 5 This is the replication kinetic curve of the recombinant turkey herpesvirus strain H20501 on CEF cells;

[0047] Figure 6 This is the result of PCR test on genetic stability of recombinant turkey herpesvirus strain H20501;

[0048] Figure 7 These are the results of indirect immunofluorescence test of the 20th generation recombinant turkey herpesvirus strain H20501 expressing F, gB, gD, and gI proteins.

[0049] Figure 8 The PCR identification results of the recombinant cosmids H483-53-F2AGB, H361-65-IBDVC2, and H220-88-GDGI;

[0050] Fig. 9 The plaque lesions produced by the recombinant turkey herpesvirus strain H30301 on CEF;

[0051] Fig.10 This is the result of PCR identification of the genomic DNA of the recombinant turkey herpesvirus strain H30301;

[0052] Fig.11 The results of the expression test of F, VP2, gB, gD, and gI proteins in CEF infected with the recombinant turkey herpesvirus strain H30301;

[0053] Fig.12 This is the replication kinetic curve of the recombinant turkey herpesvirus strain H30301 on CEF cells;

[0054] Fig.13 This is the result of PCR test on genetic stability of recombinant turkey herpesvirus strain H30301;

[0055] Fig.14 These are the results of indirect immunofluorescence tests of the 20th generation H30301 expressing F, VP2, gB, gD, and gI proteins. DETAILED DESCRIPTION

[0056] 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.

[0057] Example 1: Construction and identification of recombinant HVT expressing NDV F gene and ILTV gB, gD, gI genes

[0058] 1.1 Establishment of the multi-fragment cosmid rescue system of HVTFC126 strain

[0059] 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 CopyControlFosmid Library Production Kit, the HVTFC126 strain genomic DNA was cloned into the pCC1Fos vector. According to the results of the recombinant cosmid terminal sequencing, 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that cloned the HVTFC126 strain genomic DNA fragments and could be spliced ​​to cover the complete HVT genome were selected. Among them, HVT01 contains a nucleotide fragment of HVT FC126 genome 1-30189, HVT02 contains a nucleotide fragment of HVT FC126 genome 21572-61476, HVT03 contains a nucleotide fragment of HVT FC126 genome 50871-90478, HVT04 contains a nucleotide fragment of HVT FC126 genome 73762-108139, HVT05 contains a nucleotide fragment of HVT FC126 genome 96776-135815, and HVT06 contains a nucleotide fragment of HVT FC126 genome 129602-159160. The above 6 recombinant cosmids cloned with HVT FC126 genomic DNA fragments were extracted and co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. Cytopathic effects can be observed 4-5 days after transfection, indicating that the parental virus strain HVT FC126 was rescued.

[0060] 1.2 Construction and identification of recombinant expression plasmids expressing NDVF gene and ILTV gB gene

[0061] According to the F gene coding region sequence of genotype VII NDV GM strain (GenBank accession number DQ486859), the porcine teschovirus 2A (P2A) self-cleavage peptide coding sequence was added to its 3' end, and the NDV F gene (shown in SEQ ID NO.1) was synthesized by chicken codon optimization design. According to the ILTV WG strain genome sequence (GenBank accession number JX458823), the ILTVgB gene (shown in SEQ ID NO.2) was synthesized after chicken codon optimization design. The target gene fragment F2AGB (shown in SEQ ID NO.3) containing the F and gB gene coding sequences was obtained by fusion PCR amplification using primers NDP1F, NDLTP1R, NDLTP2F and LTP2R (Table 1), wherein the F gene and gB gene coding regions are connected by the self-cleavage peptide P2A coding sequence. The target gene PCR product 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 F2AGB and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-F2AGB that co-expresses the NDVF gene and the ILTVgB gene, wherein the F2AGB gene expression framework is named CAGW-F2AGB, which sequentially contains the chicken β-actin promoter, the F gene coding sequence, the P2A coding sequence, the gB gene coding sequence, the woodchuck hepatitis virus post-transcriptional regulatory sequence and the 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 F2AGB obtained was shown in SEQ ID NO.3, which was consistent with expectations, indicating that the recombinant plasmid was constructed correctly.

[0062] 1.3 Construction and identification of recombinant cosmids expressing NDVF gene and ILTV gB gene

[0063] According to the instructions of the Counter Selection BAC Modification Kit, the F2AGB gene expression framework CAGW-F2AGB (shown in SEQ ID NO.4) was cloned into the 95322-95323 nucleotides of the HVT FC126 genome in the recombinant cosmid HVT04 using the Red / ET recombination method to construct the recombinant cosmid HVT04-53-F2AGB that co-expresses the F 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 HVT04-53-F2AGB 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 gB gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The parental cosmid HVT04 had no target gene insertion sequence, and the PCR result was negative. The above results showed that the recombinant cosmid HVT04-53-F2AGB was constructed correctly.

[0064] 1.4 Construction and identification of recombinant cosmids expressing ILTVgD and gI genes

[0065] According to the genome sequence of ILTVWG strain (GenBank accession number JX458823), ILTV gD and gI gene expression framework ILTV-GDGI (SEQ ID NO.5) was amplified by PCR. According to the instructions of the Counter Selection BAC Modification Kit, the nucleotides 140080-140729 of the HVT FC126 genome in the recombinant cosmid HVT06 were replaced with the above expression framework ILTV-GDGI by the Red / ET recombination method to construct the recombinant cosmid HVT06-88-GDGI expressing ILTVgD and gI genes. The target gene primer ILTVgIF in Table 1 and the downstream homology arm primer HVT88R of the target gene were used to identify whether the target gene expression framework was inserted into the recombinant cosmid HVT06-88-GDGI by PCR, and the PCR product was sequenced and identified. The results showed that a PCR fragment of about 1700 bp was obtained ( Figure 1 ), the size was consistent with the expectation. The recombinant cosmid was PCR amplified and sequenced using the primers HVT88F and HVT88R in Table 1. The sequencing results showed that the ILTVgD 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 HVT06-88-GDGI was constructed correctly.

[0066] Table 1 PCR primers used to construct and identify recombinant cosmids

[0067]

[0068] 1.3 Rescue and identification of recombinant HVT expressing NDV F gene and ILTV gB, gD, gI genes

[0069] The recombinant cosmids HVT04-53-F2AGB, HVT06-88-GDGI and other parental cosmids HVT01, HVT02, HVT03, and HVT05 cloned with HVT FC126 strain genomic 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 when plaque lesions appeared, and was continuously passaged and preserved in CEF cells to rescue the recombinant turkey herpes virus strain with the CAGW-F2AGB expression framework inserted between nucleotides 95322-95323 of the HVT genome and the ILTV-GDGI expression framework inserted between nucleotides 140079-140730 of the HVT genome. The strain was named H20501. The plaque lesions produced by the above recombinant turkey herpes virus strain H20501 on CEF are shown in the following figure. Figure 2 shown.

[0070] The genomic DNA of the recombinant turkey herpes virus strain H20501 was extracted, and the genomic DNA of the parent virus strain HVT FC126 was set as a control. The recombinant turkey herpes virus strain H20501 was identified by PCR using the target gene specific primer LT1348F and the target gene downstream homology arm primer HVT54R (Table 1). The results showed that the recombinant turkey herpes virus strain H20501 could be amplified to obtain a fragment of about 2700 bp, which was consistent with the expected size ( Figure 3 ). The PCR product was sequenced and found to contain the gB gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The ILTVgI gene-specific primer ILTVgIF in Table 1 and the downstream homology arm primer HVT88R of the target gene GDGI were used to perform PCR identification on the genomic DNA of the recombinant turkey herpes virus strain, and the result was amplified to obtain a PCR fragment of about 1700 bp ( Figure 3 ), the size was consistent with the expectation. The parent virus HVT FC126 strain had no target gene inserted, and the PCR result was negative. The above results showed that the target genes F2AGB and GDGI were correctly inserted into the genome of HVT FC126 strain, and the recombinant turkey herpes virus strain H20501 was correctly constructed.

[0071] Example 2: In vitro biological characteristics analysis of recombinant turkey herpesvirus expressing NDVF gene and ILTVgB, gD, gI genes

[0072] 2.1 Detection of NDVF and ILTV gB, gD, and gI proteins expressed by recombinant turkey herpesvirus strain H20501

[0073] The recombinant turkey herpes virus strain H20501 and the parent virus HVT FC126 strain were inoculated into CEF cells cultured in 6-well plates, with each virus strain inoculated into 4 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 F, gB, gD, and gI proteins was detected by indirect immunofluorescence test using NDV positive serum and gB, gD, and gI polyclonal antibodies as primary antibodies, FITC-labeled rabbit anti-chicken IgG and FITC-labeled goat anti-rabbit IgG as secondary antibodies, and the parent virus HVT FC126 strain-infected CEF was used as a negative control. The results showed that the cells infected with the recombinant turkey herpes virus strain H20501 could react with NDV positive serum and gB, gD, and gI polyclonal 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 turkey herpesvirus strain H20501 can co-express NDVF protein and ILTV gB, gD, and gI proteins in infected cells.

[0074] 2.2 Analysis of in vitro replication characteristics of recombinant turkey herpesvirus strain H20501

[0075] The recombinant turkey herpes virus strain H20501 and the parental virus HVT FC126 strain 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 turkey herpes virus strain H20501 and the parental virus HVT FC126 strain in CEF. The results showed that the replication titers of the recombinant turkey herpes virus strain H20501 and the parental virus HVT FC126 strain reached the highest peak 120 hours after infection, which were 1.92×10 5 PFU / ml and 2.18×10 5 PFU / ml, the titer of the recombinant turkey herpesvirus strain H20501 at each time point after infection with CEF was not significantly different from that of the parent virus HVT FC126 strain (P>0.05)( Figure 5 The above results showed that the recombinant turkey herpesvirus strain H20501 had good replication ability on CEF and its in vitro replication characteristics were consistent with those of the parent virus H20501.

[0076] 2.3 Genetic stability test of recombinant turkey herpesvirus strain H20501

[0077] The rescued recombinant turkey herpes virus strain H20501 was continuously passaged on CEF to the 20th generation, and the genomic DNA of the 20th generation recombinant turkey herpes virus strain was extracted for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the genome of the recombinant turkey herpes virus strain H20501. The results showed that the PCR identification of the 20th generation recombinant turkey herpes virus strain using the target gene F2AGB specific primer LT1348F and the target gene downstream homology arm primer HVT54R (Table 1) can amplify a fragment of about 2700bp, which is consistent with the expected size ( Figure 6 ). The genomic DNA of the recombinant turkey herpes virus strain was amplified and sequenced by PCR using primers CAGF and CAGR, respectively, and it was found that the sequence of the target gene F2AGB inserted into the genome of the HVT FC126 strain was correct. The genomic DNA of the 20th generation recombinant turkey herpes virus strain was identified by PCR using the target gene GDGI specific primer ILTVgIF and the target gene downstream homology arm primer HVT88R (Table 1). As a result, a PCR fragment of about 1700 bp was amplified ( Figure 6 ), the size was correct. At the same time, the genomic DNA of the recombinant turkey herpesvirus strain was amplified by PCR and sequenced using primers HVT88F and HVT88R (Table 1), and the results showed that the ILTVgD and gI gene expression framework sequences inserted into the genome of the HVT FC126 strain were correct.

[0078] At the same time, the 20th generation recombinant turkey herpes virus strain was inoculated into CEF cells, and indirect immunofluorescence tests were performed using NDV positive serum and gB, gD, and gI polyclonal antibodies to detect the stability of target gene expression. The results showed that after the recombinant turkey herpes virus strain H20501 was continuously propagated on CEF cells to the 20th generation, the exogenous target genes F, gB, gD, and gI could still be stably expressed ( Figure 7 The above results show that the NDVF gene and ILTV gB, gD, and gI genes inserted into the genome of the HVT FC126 strain can stably exist during the virus passage process, and the recombinant turkey herpes virus strain H20501 has good genetic stability.

[0079] Example 3: Safety and immunogenicity testing of recombinant turkey herpesvirus strain H20501

[0080] 3.1 Safety testing of recombinant turkey herpesvirus strain H20501

[0081] The fifth-generation recombinant turkey herpes virus strain H20501 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 weights were weighed to evaluate the effect of the recombinant turkey herpes virus strain H20501 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the bursa of Fabricius, thymus, spleen, liver and other organs were collected, weighed, and observed for atrophy or swelling symptoms. The results showed that the recombinant turkey herpes virus strain H20501 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 the weight of the chickens in the recombinant turkey herpes virus strain H20501 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 indicate that the recombinant turkey herpesvirus strain H20501 is safe for SPF chickens.

[0082] 3.2 Immunoprotection test of recombinant turkey herpesvirus strain H20501 against NDV and ILTV

[0083] 60 one-day-old SPF chicks were randomly divided into 3 groups, 20 in each group. Group 1 was subcutaneously inoculated with the recombinant turkey herpes virus strain H20501 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parent virus HVT FC126 strain at the same dose, and Group 3 was not immunized as a blank control group. 28 days after immunization, 10 chickens in Group 1 and Group 2 were respectively challenged with the virulent GM strain of NDV of type VII gene, and observed for 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. The remaining 10 chickens in Group 1 and Group 2 were respectively challenged with the virulent WG strain of ILTV; observed for 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. Group 3 was not challenged and served as a healthy control group.

[0084] The results showed that all 10 chickens in the parental virus HVT FC126 inoculation group died after being challenged with NDV, while all 10 chickens in the recombinant turkey herpes virus H20501 inoculation group survived after being challenged with NDV without obvious clinical symptoms. After the parental virus FC126 inoculation group was challenged with ILTV, all 10 experimental chickens showed symptoms such as lethargy, ruffled feathers, dyspnea, conjunctivitis, head shaking, and tearing, of which 6 died; autopsy showed that the affected chickens had laryngeal and tracheal bleeding, cheesy exudates and other lesions. After the 10 chickens in the recombinant turkey herpes virus H20501 inoculation group were challenged with ILTV, one chicken showed mild symptoms of lethargy, and laryngeal hemorrhage lesions were found in autopsy, while the other 9 chickens did not show adverse clinical symptoms or autopsy lesions. The above results showed that the recombinant turkey herpesvirus strain H20501 could provide 100% and 90% immune protection rates against NDV and ILTV virulent attacks, respectively.

[0085] Example 4: Construction and identification of recombinant HVT expressing NDV F gene, IBDV VP2 gene and ILTV gB, gD, gI genes

[0086] 4.1 Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0087] 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 LibraryProduction Kit, the HVTFC126 strain genomic DNA was cloned into the pCC1Fos vector. The constructed recombinant cosmid was terminally sequenced with primers pCC1F and pCC1R (see Table 2 for details). According to the sequencing results, 6 recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned HVT FC126 strain genomic DNA fragments and could be spliced ​​to cover the complete HVT genome were selected. Among them, H434 contains the nucleotide fragment of the HVT FC126 strain genome 1-40394, H481 contains the nucleotide fragment of the HVT FC126 strain genome 31586-69191, H159 contains the nucleotide fragment of the HVT FC126 strain genome 58293-92645, H483 contains the nucleotide fragment of the HVT FC126 strain genome 76940-110712, H361 contains the nucleotide fragment of the HVT FC126 strain genome 103006-134393, and H220 contains the nucleotide fragment of the HVT FC126 strain genome 123621-159160. The above 6 recombinant cosmids cloned with the genomic DNA fragments of the HVT FC126 strain were extracted, and the 6 recombinant cosmids were co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. Cytopathic effects can be observed 4-5 days after transfection, indicating that the parental virus strain FC126 was rescued.

[0088] 4.2 Construction and identification of recombinant cosmids expressing NDVF gene and ILTV gB gene

[0089] According to the F gene coding region sequence of genotype VII NDV GM strain (GenBank accession number DQ486859), the porcine teschovirus 2A (P2A) self-cleavage peptide coding sequence was added to its 3' end, and the NDV F gene (shown in SEQ ID NO.1) was synthesized by chicken codon optimization design. According to the ILTV WG strain genome sequence (GenBank accession number JX458823), the ILTVgB gene (shown in SEQ ID NO.2) was synthesized after chicken codon optimization design. The target gene fragment F2AGB (shown in SEQ ID NO.3) containing the F and gB gene coding sequences was obtained by fusion PCR amplification using primers NDP1F, NDLTP1R, NDLTP2F and LTP2R (Table 2), wherein the F gene and gB gene coding regions are connected by the self-cleavage peptide P2A coding sequence. The target gene PCR product 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 F2AGB and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-F2AGB that co-expresses the NDVF gene and the ILTVgB gene, wherein the F2AGB gene expression framework CAGW-F2AGB sequentially includes the chicken β-actin promoter, the F gene coding sequence, the P2A coding sequence, the gB gene coding sequence, the woodchuck hepatitis virus post-transcriptional regulatory sequence and the rabbit β-globulin polyadenylation sequence. The recombinant plasmid was sequenced using primers CAGF and CAGR (Table 2), and the nucleotide sequence of the target gene F2AGB obtained was shown in SEQ ID NO.3, which was consistent with expectations, indicating that the recombinant plasmid was constructed correctly.

[0090] According to the instructions of the Counter Selection BAC Modification Kit, the F2AGB gene expression framework CAGW-F2AGB (shown in SEQ ID NO.4) was cloned into the recombinant cosmid H483 between nucleotides 95322-95323 of the HVT FC126 strain genome using the Red / ET recombination method to construct the recombinant cosmid H483-53-F2AGB that co-expresses the F gene and gB gene. The target gene-specific primer LT1348F and the target gene downstream homology arm primer HVT54R (Table 2) were used to identify whether the recombinant cosmid H483-53-F2AGB was inserted into the target gene expression framework by PCR. The results showed that the PCR fragment of about 2700 bp was obtained ( Figure 8), 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 parental cosmid H483 had no target gene insertion sequence, and the PCR result was negative. The above results showed that the recombinant cosmid H483-53-F2AGB was constructed correctly.

[0091] 4.3 Construction and identification of recombinant cosmids expressing ILTVgD and gI genes

[0092] According to the genome sequence of ILTVWG strain (GenBank accession number JX458823), ILTV gD and gI gene expression framework ILTV-GDGI (SEQ ID NO.5) was amplified by PCR. According to the instructions of the Counter Selection BAC Modification Kit, the nucleotides 140080-140729 of the HVT FC126 strain genome in the recombinant cosmid H220 were replaced with the above expression framework ILTV-GDGI by the Red / ET recombination method to construct the recombinant cosmid H220-88-GDGI expressing ILTVgD 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 a PCR fragment of about 1700 bp was obtained ( Figure 8 ), the size was consistent with the expectation. 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 genome of the HVT FC126 strain, and the target gene sequence was correct. The above results indicate that the recombinant cosmid H220-88-GDGI was constructed correctly.

[0093] 4.4 Construction and identification of recombinant cosmids expressing IBDV VP2 gene

[0094] According to the VP2 gene sequence of IBDVFaragher 52 / 70 strain (GenBank accession number HG974565), the target gene IBDVVP2 (shown in SEQ ID NO.6) was synthesized. The recombinant plasmid pUC57-IBDVC2 cloned with the IBDVVP2 gene was used as a template, and the IBDVVP2 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 the recombinant plasmid pCMV-IBDVC2 expressing the IBDVVP2 gene.

[0095] According to the instructions of the Counter Selection BAC Modification Kit, the IBDVVP2 gene expression framework CMV-IBDVC2 (containing the mouse cytomegalovirus promoter, the IBDV classic strong strain VP2 gene and the SV40 polyadenylation sequence, as shown in SEQ ID NO.7) was cloned into the recombinant cosmid H361 between nucleotides 112071-112088 of the HVT FC126 strain genome using the Red / ET recombination method to construct the recombinant cosmid H361-65-IBDVC2 expressing the IBDVVP2 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 8 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the IBDVVP2 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.

[0096] Table 2 PCR primers used to construct and identify recombinant cosmids

[0097]

[0098] 4.5 Rescue and identification of recombinant HVT expressing NDV F gene, IBDV VP2 gene and ILTV gB, gD, gI genes

[0099] The recombinant cosmids H483-53-F2AGB, H361-65-IBDVC2, H220-88-GDGI and other parental cosmids H434, H481, and H159 cloned with HVT 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 plaque lesions appeared after 4-5 days of culture, and was continuously passaged and preserved in CEF cells to rescue the recombinant turkey herpes virus strain in which the NDVF gene, IBDVVP2 gene, and ILTVgB, gD, and gI genes were simultaneously inserted into the HVT genome. The strain was named H30301. The plaque lesions produced by the above recombinant turkey herpes virus strain on CEF are shown in the figure. Fig. 9 shown.

[0100] The genomic DNA of the recombinant turkey herpes virus strain H30301 was extracted, and the genomic DNA of the parent virus strain HVT FC126 was set as a control. The recombinant turkey herpes virus strain was identified by PCR using the target gene F2AGB specific primer LT1348F and the downstream homology arm primer HVT54R (Table 2). The results showed that the recombinant turkey herpes virus strain H30301 amplified a fragment of about 2700 bp, which was consistent with expectations ( Fig.10 ). The recombinant turkey herpesvirus strain was identified by PCR using the IBDVVP2 gene specific primer IBDVC2F and the downstream homology arm primer HVT65F. The results showed that the recombinant turkey herpesvirus strain H30301 amplified a PCR fragment of about 1800 bp ( Fig.10 ), which is consistent with expectations. The ILTV gI specific primer ILTVgIF and the downstream homology arm primer HVT88R were used to perform PCR identification on the genomic DNA of the recombinant turkey herpesvirus strain, and a PCR fragment of about 1700 bp was obtained ( Fig.10 ), which was consistent with expectations. The parent virus HVT FC126 strain had no target gene insertion, and the PCR result was negative. The above results showed that the target genes F2AGB, IBDVVP2 and GDGI were correctly inserted into the genome of HVT FC126 strain, and the recombinant turkey herpes virus strain H30301 was correctly constructed.

[0101] Example 5: In vitro biological characteristics analysis of recombinant turkey herpesvirus expressing NDVF gene, IBDVVP2 gene and ILTV gB, gD, gI genes

[0102] 5.1 Detection of recombinant turkey herpesvirus strains expressing NDVF, IBDVVP2, and ILTV gB, gD, and gI proteins

[0103] The recombinant turkey herpes virus strain H30301 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 NDV positive serum, VP2 monoclonal antibody, and gB, gD, and gI polyclonal antibodies were used as primary antibodies, and FITC-labeled rabbit anti-chicken IgG, FITC-labeled goat anti-mouse IgG, and FITC-labeled goat anti-rabbit IgG were used as secondary antibodies. The expression of F, VP2, gB, gD, and gI proteins was detected by indirect immunofluorescence assay, and CEF inoculated with the parental virus HVT FC126 strain was used as a negative control. The results showed that cells infected with the recombinant turkey herpes virus strain H30301 could react with NDV positive serum, VP2 monoclonal antibody, and gB, gD, and gI polyclonal antibodies, showing green fluorescence signals ( Fig.11). No fluorescence was observed in cells infected with the parent virus HVTFC126 strain and in control cells that were not infected. The above results indicate that the recombinant turkey herpesvirus strain H30301 can co-express F, VP2, gB, gD, and gI proteins in infected cells.

[0104] 5.2 Analysis of in vitro replication characteristics of recombinant turkey herpesvirus strain H30301

[0105] The recombinant turkey herpes virus strain H30301 and the parental virus HVT FC126 strain 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 turkey herpes virus strain H30301 and the parental virus HVT FC126 strain in CEF. The results showed that the replication titers of the recombinant turkey herpes virus strain H30301 and the parental virus HVT FC126 strain reached the highest peak 120 hours after infection, which were 1.82×10 5 PFU / ml and 1.95×10 5 PFU / ml, the titer of the recombinant turkey herpes virus strain at each time point after infection with CEF was not significantly different from that of the parent virus (P>0.05)( Fig.12 ). The above results show that the recombinant turkey herpesvirus strain H30301 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus HVT FC126 strain.

[0106] 5.3 Genetic stability test of recombinant turkey herpesvirus strain H30301

[0107] The rescued recombinant turkey herpes virus strain H30301 was continuously passaged on CEF to the 20th generation, and the genomic DNA of the 20th generation recombinant turkey herpes virus strain was extracted for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the genome of the recombinant turkey herpes virus strain. The results showed that the PCR identification of the 20th generation recombinant turkey herpes virus strain using the target gene F2AGB specific primer LT1348F and the downstream homology arm primer HVT54R (Table 2) could amplify a fragment of about 2700bp, which was consistent with expectations ( Fig.13); The genomic DNA of the recombinant turkey herpes virus strain was amplified and sequenced using primers CAGF and CAGR, and the sequence of the target gene F2AGB inserted into the HVT genome was found to be correct. The 20th generation recombinant turkey herpes virus strain 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 ( Fig.13 ), which was consistent with expectations; sequencing results showed that the PCR product contained the IBDVVP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The 20th generation recombinant turkey herpes virus strain 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 ( Fig.13 ), the size was correct; the genomic DNA of the recombinant turkey herpesvirus strain was amplified by PCR and sequenced using primers HVT88F and HVT88R (Table 2), and the results showed that the ILTV gD and gI gene expression framework sequences inserted into the HVT FC126 strain genome were correct.

[0108] At the same time, the 20th generation recombinant turkey herpes virus strain was inoculated into CEF cells, and indirect immunofluorescence tests were performed using NDV positive serum, VP2 monoclonal antibody, and gB, gD, and gI polyclonal antibodies to detect the stability of target gene expression. The results showed that after the recombinant turkey herpes virus strain H30301 was continuously propagated on CEF cells to the 20th generation, the exogenous target genes F, VP2, gB, gD, and gI could still be stably expressed ( Fig.14 ). The above results show that the F, VP2, gB, gD, and gI genes inserted into the HVT genome can all stably exist during the virus passage process, and the recombinant turkey herpes virus strain H30301 has good genetic stability.

[0109] Example 6: Safety and immunogenicity testing of recombinant turkey herpesvirus strain H30301

[0110] 6.1 Safety testing of recombinant turkey herpesvirus strain H30301

[0111] The fifth-generation recombinant turkey herpes virus strain H30301 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 turkey herpes virus strain H30301 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the bursa of Fabricius, thymus, spleen, liver and other organs were collected, weighed, and observed for atrophy or swelling symptoms. The results showed that the recombinant turkey herpes virus strain H30301 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 the weight of the chickens in the group inoculated with the recombinant turkey herpes virus strain H30301 was not 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 indicate that the recombinant turkey herpesvirus strain H30301 is safe for SPF chickens.

[0112] 6.2 Immunoprotection test of recombinant turkey herpesvirus strain H30301 against NDV

[0113] 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 turkey herpes virus strain H30301 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parental virus HVT FC126 strain 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 virulent GM strain of NDV respectively; 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 of the test chickens were recorded. The results showed that all 10 chickens in the parental virus FC126 inoculation group became ill and died after being challenged with NDV, while all 10 chickens in the recombinant turkey herpes virus strain H30301 inoculation group survived healthily after being challenged with NDV, without obvious clinical symptoms.

[0114] 6.3 Immunoprotection test of recombinant turkey herpesvirus strain H30301 against IBDV super-virulent virus

[0115] 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 turkey herpes virus strain H30301 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parent virus HVT FC126 strain 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 strain inoculation group was challenged with IBDV super-virulent virus, all 10 chickens showed symptoms such as lethargy, ruffled feathers, and inability to lie on the ground. Nine of the sick chickens died, and autopsy showed atrophy, streaky hemorrhages, or yellow jelly-like infiltration lesions in the bursa of Fabricius of all 10 chickens. All 10 chickens in the recombinant turkey herpesvirus strain H30301 inoculation group were healthy and survived after being challenged with IBDV super-virulent virus, and autopsy found no obvious lesions in the bursa of Fabricius.

[0116] 6.4 Immunoprotection test of recombinant turkey herpesvirus strain H30301 against ILTV

[0117] 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 turkey herpes virus strain H30301 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parental virus HVT FC126 strain 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 ILTV WG strain 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 group vaccinated with the recombinant turkey herpesvirus strain H30301 were challenged with the strong ILTV virus, one chicken showed symptoms of mental depression and dyspnea, and laryngeal hemorrhagic lesions were found in the autopsy. The other 9 chickens did not show adverse clinical symptoms or autopsy lesions.

Claims

1. A recombinant turkey herpesvirus strain (Herpesvirus of turkey, HVT) that co-expresses the Newcastle disease virus (NDV) F gene and the infectious laryngotracheitis virus (ILTV) gB, gD, and gI genes, characterized in that: The recombinant turkey herpesvirus strain is obtained by inserting the expression frame CAGW-F2AGB containing NDV F gene and ILTV gB gene and the expression frame ILTV-GDGI containing ILTVgD and gI genes into the turkey herpesvirus genome.

2. The recombinant turkey herpesvirus strain that co-expresses Newcastle disease virus F gene and infectious laryngotracheitis virus gB, gD, gI genes as claimed in claim 1, characterized in that: The turkey herpes virus is HVT FC126 strain, and the GenBank accession number of the HVTFC126 strain genome DNA sequence is AF291866.

3. The recombinant turkey herpesvirus strain that co-expresses Newcastle disease virus F gene and infectious laryngotracheitis virus gB, gD, gI genes as claimed in claim 2, characterized in that: The expression framework CAGW-F2AGB is inserted between nucleotides 95322-95323 of the genome of HVT FC126 strain; the expression framework ILTV-GDGI is inserted between nucleotides 140079-140730 of the genome of HVT FC126 strain.

4. The recombinant turkey herpesvirus strain that co-expresses Newcastle disease virus F gene and infectious laryngotracheitis virus gB, gD, gI genes as claimed in claim 3, characterized in that: The expression frame CAGW-F2AGB comprises a chicken β-actin promoter, NDVF gene and ILTV gB gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylic acid sequence, wherein the NDVF gene and ILTVgB gene coding sequences are connected by a porcine teschovirus 2A (P2A) self-cleavage peptide coding sequence; preferably, the nucleotide sequence of the expression frame CAGW-F2AGB is shown in SEQ ID NO.4, and preferably, the nucleotide sequence of the expression frame ILTV-GDGI is shown in SEQ ID NO.

5.

5. A recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene, the infectious bursal disease virus (IBDV) VP2 gene, and the infectious laryngotracheitis virus gB, gD, and gI genes, characterized in that: The recombinant turkey herpesvirus strain is obtained by further inserting the expression framework CMV-IBDVC2 containing the IBDVVP2 gene into the turkey herpesvirus genome on the basis of the recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene and the infectious laryngotracheitis virus gB, gD, and gI genes as described in any one of claims 1 to 4.

6. The recombinant turkey herpesvirus strain that co-expresses Newcastle disease virus F gene, infectious bursal disease virus VP2 gene, and infectious laryngotracheitis virus gB, gD, gI genes as claimed in claim 5, characterized in that: The expression frame CMV-IBDVC2 comprises a mouse cytomegalovirus promoter, an IBDV classic virulent strain VP2 gene and an SV40 polyadenylation sequence, and its nucleotide sequence is shown in SEQ ID NO.

7. Preferably, the expression frame CMV-IBDVC2 is inserted between nucleotides 112071-112088 of the HVT FC126 strain genome.

7. A method for constructing a recombinant turkey herpesvirus strain that co-expresses the Newcastle disease virus F gene and the infectious laryngotracheitis virus gB, gD, and gI genes as described in 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 The genomic DNA of HVT FC126 strain was extracted and cloned into the pCC1Fos vector in segments. Six recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05 and HVT06, which cloned the genomic DNA fragments of HVT FC126 strain and could be assembled to cover the complete genome of HVT FC126 strain, were selected; among them, HVT01 contained the nucleotide fragment of 1-30189 of the genome of HVT FC126 strain, HVT02 contained the nucleotide fragment of 21572-61476 of the genome of HVT FC126 strain, HVT03 contained the nucleotide fragment of 50871-90478 of the genome of HVTFC126 strain, HVT04 contained the nucleotide fragment of 73762-108139 of the genome of HVT FC126 strain, and HVT05 contained the nucleotide fragment of 50871-90478 of the genome of HVTFC126 strain. The nucleotide fragment of the FC126 strain genome is from 96776 to 135815, and HVT06 contains the nucleotide fragment of the HVTFC126 strain genome from 129602 to 159160; (2) Construction of recombinant cosmids expressing NDVF and ILTVgB genes A recombinant plasmid comprising a chicken β-actin promoter, NDVF 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-F2AGB was constructed, wherein the F gene and gB gene coding regions were connected by a P2A self-cleaving peptide coding sequence; the expression frame CAGW-F2AGB was cloned into the 95322-95323 nucleotides of the HVT FC126 strain genome in the recombinant cosmid HVT04 by using the Red / ET recombination method, and a recombinant cosmid that co-expressed the F gene and the gB gene was constructed and named HVT04-53-F2AGB; (3) 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 HVT06 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 HVT06-88-GDGI; (4) Rescue of recombinant HVT expressing NDVF gene and ILTVgB, gD, and gI genes The recombinant cosmids HVT04-53-F2AGB, HVT06-88-GDGI and the parental cosmids HVT01, HVT02, HVT03 and HVT05 cloned with the genome fragment of HVTFC126 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, and the viruses were harvested after the appearance of plaque lesions in culture, and the recombinant turkey herpes virus strain with the expression frame CAGW-F2AGB inserted between nucleotides 95322-95323 of the HVT FC126 strain genome and the expression frame ILTV-GDGI inserted between nucleotides 140079-140730 of the HVTFC126 strain genome was rescued and named H20501.

8. A method for constructing a recombinant turkey herpesvirus strain that co-expresses Newcastle disease virus F gene, infectious bursal virus VP2 gene, and infectious laryngotracheitis virus gB, gD, gI genes as described in claim 5 or 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 genome were selected; among them, H434 contained the nucleotide fragment of 1-40394 of the genome of HVT FC126 strain, H481 contained the nucleotide fragment of 31586-69191 of the genome of HVT FC126 strain, H159 contained the nucleotide fragment of 58293-92645 of the genome of HVT FC126 strain, H483 contained the nucleotide fragment of 76940-110712 of the genome of HVT FC126 strain, and H361 contained the nucleotide fragment of 58293-92645 of the genome of HVT FC126 strain. The nucleotide fragment of the FC126 strain genome is 103006-134393, and H220 contains the nucleotide fragment of the HVT FC126 strain genome at 123621-159160; (2) Construction of recombinant cosmids expressing NDVF and ILTVgB genes A recombinant plasmid comprising a chicken β-actin promoter, NDVF 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-F2AGB is constructed, wherein the F gene and gB gene coding regions are connected by a P2A self-cleaving peptide coding sequence; the expression frame CAGW-F2AGB 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 F gene and the gB gene is constructed and named H483-53-F2AGB; (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 NDV F gene, IBDV VP2 gene, and ILTV gB, gD, and gI genes The recombinant cosmids H483-53-F2AGB, H361-65-IBDVC2, H220-88-GDGI and the parental cosmids H434, H481, and H159 cloned with the HVT FC126 strain genome fragment 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, and the viruses were harvested after the appearance of plaque lesions in culture, and the recombinant HVT with NDV F gene, IBDVVP2 gene, and ILTV gB, gD, and gI genes inserted into the HVTFC126 strain genome was rescued and named H30301.

9. 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 Newcastle disease and infectious laryngotracheitis.

10. Use of the recombinant turkey herpesvirus strain according to claim 5 or 6 in the preparation of a vaccine for simultaneously preventing Newcastle disease, infectious bursal disease and infectious laryngotracheitis.

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