Recombinant herpesvirus turkey capable of co-expressing H9 subtype AIVHA gene and ILTV gB, gD and gI genes and application of recombinant herpesvirus turkey

Abstract

The invention discloses a recombinant herpesvirus turkey strain for co-expressing an H9 subtype AIVHA 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 ILTVgD and gI genes and parent clay containing HVT genome DNA fragments and then carrying out virus rescue. The recombinant herpesvirus of turkeys is prepared by the following steps: cotransfecting CEF cells with recombinant clay containing H9 subtype AIVHA genes and ILTVgB 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 SPF chickens are immunized, the SPF chickens can obtain immune protection against H9 subtype AIV and ILTV virulent viruses. The invention provides a new technical means for simultaneously preventing the H9 subtype avian influenza and the avian infectious laryngotracheitis.

Description

Technical Field

[0001] The invention relates to a recombinant turkey herpes virus which co-expresses the HA gene of H9 subtype avian influenza virus and the gB, gD and gI genes of infectious laryngotracheitis virus of chickens and the application of the recombinant turkey herpes virus in preparing vaccines of H9 subtype avian influenza and infectious laryngotracheitis of chickens, 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. It is now distributed worldwide. Its widespread prevalence in poultry poses a continuous threat to the global poultry industry and public health security. Since the H9 subtype AIV was first isolated in Guangdong Province in 1992, the disease has been prevalent in my country for more than 30 years. Broilers are the main host of the H9 subtype AIV. In recent years, the virus has continued to maintain a high level of prevalence in my country and has become one of the most serious pathogens affecting the development of my country's broiler farming industry. In particular, when mixed with pathogens such as infectious bronchitis virus (IBV), broilers suffer from severe bronchial obstruction symptoms, which seriously affects the health of the flock. AIV belongs to the Orthomyxoviridae family. The HA protein it encodes can recognize specific receptors and has strong immunogenicity. It is the main host protective antigen of the virus and can induce the production of neutralizing antibodies. The H9 subtype avian influenza is currently mainly prevented and controlled through inactivated vaccines. However, in recent years, the isolation rate of H9 subtype AIV in my country has increased year by year, indicating that traditional inactivated vaccine immunization can no longer effectively prevent and control the virus infection. There is an urgent need to develop a safer and more efficient new vaccine.

[0003] Infectious 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] 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

[0005] The purpose of the present invention is to provide a vaccine strain capable of preventing H9 subtype avian influenza and infectious laryngotracheitis of chickens and its application.

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

[0007] The vaccine strain capable of simultaneously preventing H9 subtype avian influenza and infectious laryngotracheitis of chickens is a recombinant turkey herpes virus that co-expresses the H9 subtype AIVHA gene and the ILTV gB, gD and gI genes. The recombinant turkey herpes virus strain is obtained by inserting the expression frame CAGW-HAGB containing the H9 subtype AIVHA gene and the ILTV gB gene and the expression frame ILTV-GDGI containing the ILTV gD and gI genes into the HVT genome.

[0008] Among them, preferably, the expression framework CAGW-HAGB containing the H9 subtype AIVHA gene and the ILTVgB gene is inserted between nucleotides 95322-95323 or between nucleotides 112071-112088 of the HVT FC126 strain genome, and the GenBank accession number of the HVT FC126 strain genomic DNA sequence is AF291866.

[0009] Preferably, the expression framework ILTV-GDGI comprising ILTVgD and gI genes is inserted between nucleotides 140079-140730 of the HVTFC126 genome, and the GenBank accession number of the HVT FC126 genome DNA sequence is AF291866.

[0010] Among them, preferably, the expression framework CAGW-HAGB containing the H9 subtype AIVHA gene and ILTVgB gene comprises in sequence: chicken β-actin promoter-HA gene coding sequence-swine teschovirus 2A self-cleavage peptide coding sequence-gB gene coding sequence-woodchuck hepatitis virus post-transcriptional regulatory sequence-rabbit β-globulin polyadenylic acid sequence.

[0011] Among them, preferably, the nucleotide sequence of the expression framework CAGW-HAGB is shown in SEQ ID NO.4.

[0012] Preferably, the nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.5.

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

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

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

[0016] (2) Construction of recombinant cosmids expressing H9 subtype AIVHA gene and ILTV gB gene

[0017] A recombinant plasmid of an expression framework CAGW-HAGB, which sequentially comprises a chicken β-actin promoter, an HA gene coding sequence, a porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence, a gB gene coding sequence, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence, is constructed; the expression framework CAGW-HAGB is cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 or the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 by using the Red / ET recombination method, to construct a recombinant cosmid that co-expresses the HA gene and the gB gene, which is named HVT04-53-HAGB or HVT05-65-HAGB;

[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 and 140730 of the HVT genome using the 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 H9 subtype AIV HA gene and ILTV gB, gD, and gI genes

[0021] The recombinant cosmids HVT04-53-HAGB, HVT05-65-HAGB, HVT06-88-GDGI and the parental cosmids HVT01, HVT02, HVT03, HVT04, and HVT05 cloned with the HVT FC126 strain genome fragment 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 viruses in which the expression framework CAGW-HAGB was inserted between nucleotides 95322-95323 or 112071-112088 of the HVT FC126 strain genome, and the expression framework ILTV-GDGI was inserted between nucleotides 140079-140730 of the HVT FC126 strain genome were rescued and named H20203 or H20204, respectively.

[0022] Among them, preferably, the nucleotide sequence of the expression framework CAGW-HAGB is shown in SEQ ID NO.4.

[0023] Preferably, the nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.5.

[0024] Finally, the present invention also proposes the use of the recombinant turkey herpes virus strain in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza and infectious laryngotracheitis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a vaccine strain that can be used to prevent H9 subtype avian influenza and infectious laryngotracheitis at the same time. The vaccine strain is obtained by inserting an expression frame CAGW-HAGB containing H9 subtype AIV HA gene and ILTV gB gene and an expression frame ILTV-GDGI containing ILTV gD and gI genes into the HVT genome. The recombinant virus strain obtained by the present invention has good in vitro replication ability and genetic stability. After immunizing SPF chickens, the SPF chickens can obtain immune protection against H9 subtype AIV and ILTV strong toxins. The present invention provides a new technical means for preventing H9 subtype avian influenza and infectious laryngotracheitis at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The PCR identification results of the recombinant cosmids HVT04-53-HAGB, HVT05-65-HAGB, and HVT06-88-GDGI;

[0028] Figure 2 The plaque lesions produced by the recombinant viruses H20203 and H20204 on CEF;

[0029] Figure 3 The results of PCR identification of the genomic DNA of the recombinant viruses H20203 and H20204;

[0030] Figure 4 The results of the expression test of HA, gB, gD, and gI proteins in CEF infected with recombinant viruses H20203 and H20204;

[0031] Figure 5 The replication kinetic curves of recombinant viruses H20203 and H20204 on CEF cells;

[0032] Figure 6 The results of PCR test for genetic stability of recombinant viruses H20203 and H20204;

[0033] Figure 7 These are the results of indirect immunofluorescence tests of the 20th generation H20203 and H20204 expressing HA, gB, gD, and gI proteins. DETAILED DESCRIPTION

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

[0035] Example 1: Construction and identification of recombinant HVT expressing H9 subtype AIVHA gene and ILTVgB, gD, gI genes

[0036] 1.1 Establishment of the multi-fragment cosmid rescue system of HVTFC126 strain

[0037] 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 FC126 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 FC126 strain genomic DNA fragments and could be spliced ​​to cover the complete HVT genome were selected. Among them, HVT01 contains a nucleotide fragment of 1-30189 of the FC126 genome, HVT02 contains a nucleotide fragment of 21572-61476 of the FC126 genome, HVT03 contains a nucleotide fragment of 50871-90478 of the FC126 genome, HVT04 contains a nucleotide fragment of 73762-108139 of the FC126 genome, HVT05 contains a nucleotide fragment of 96776-135815 of the FC126 genome, and HVT06 contains a nucleotide fragment of 129602-159160 of the FC126 genome. The above 6 recombinant clays cloned with FC126 genomic DNA fragments were extracted, and the 6 recombinant clays were co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection. The appearance of cytopathic effects can be observed 4-5 days after transfection, that is, the parental virus strain HVT FC126 was rescued.

[0038] 1.2 Construction and identification of recombinant plasmids expressing H9AIVHA and ILTVgB genes

[0039] According to the HA gene coding region sequence of the H9 subtype AIVTJ 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 (shown in SEQ ID NO.1) was synthesized after chicken codon optimization. According to the ILTVWG 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 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 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, in order to enhance the transcription and translation efficiency of the target gene, 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 polyadenylic acid sequence to obtain the recombinant plasmid pCAGW-HAGB for co-expressing the H9 AIV HA gene and the ILTV gB gene, wherein the HAGB gene expression framework (named CAGW-HAGB) sequentially includes: chicken β-actin promoter-HA gene coding sequence-P2A coding sequence-gB gene coding sequence-woodchuck hepatitis virus post-transcriptional regulatory sequence-rabbit β-globulin polyadenylic acid sequence. 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.

[0040] 1.3 Construction and identification of recombinant cosmids expressing H9AIVHA and ILTVgB genes

[0041] 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 inserted into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 using the Red / ET recombination method to construct the recombinant cosmid HVT04-53-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 HVT04-53-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 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-HAGB was constructed correctly.

[0042] 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 inserted into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 using the Red / ET recombination method to construct the recombinant cosmid HVT05-65-HAGB that co-expresses the HA gene and gB gene. The target gene-specific primer LT1348F and the target gene downstream homology arm primer HVT65F (Table 1) were used to identify whether the recombinant cosmid HVT05-65-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 above results indicate that the recombinant cosmid HVT05-65-HAGB was constructed correctly.

[0043] 1.4 Construction and identification of recombinant cosmids expressing ILTVgD and gI genes

[0044] According to the genome sequence of ILTVWG strain (GenBank accession number JX458823), ILTV gD and gI gene expression framework ILTV-GDGI (shown in SEQ ID NO.5) was amplified by PCR. According to the instructions of Counter Selection BAC Modification Kit, the expression framework ILTV-GDGI was inserted into the HVT genome between nucleotides 140080-140729 in the recombinant cosmid HVT06 by Red / ET recombination method to construct the recombinant cosmid HVT06-88-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 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 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 genome and the target gene sequence was correct. The above results indicate that the recombinant cosmid HVT06-88-GDGI was constructed correctly.

[0045] Table 1 PCR primers used to construct and identify recombinant cosmids

[0046]

[0047]

[0048] 1.5 Rescue and identification of recombinant HVT expressing H9 subtype AIVHA gene and ILTVgB, gD, and gI genes

[0049] Recombinant cosmids HVT04-53-HAGB, HVT05-65-HAGB, HVT06-88-GDGI and other parental cosmids HVT01, HVT02, HVT03, HVT04 and HVT05 cloned with HVT genome fragments were extracted using a plasmid extraction kit. Recombinant cosmids HVT04-53-HAGB, HVT06-88-GDGI and parental cosmids HVT01, HVT02, HVT03 and HVT05 were co-transfected into CEF cells using the calcium phosphate transfection method. Viruses were harvested after 4-5 days of culture until plaque lesions appeared, and were continuously passaged and preserved in CEF cells. The recombinant virus with the CAGW-HAGB 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 was rescued and named H20203. The recombinant cosmids HVT05-65-HAGB and HVT06-88-GDGI were co-transfected with the parental cosmids HVT01, HVT02, HVT03, and HVT04 into CEF cells. After 4-5 days of culture, the virus was harvested after plaque lesions appeared. The virus was continuously passaged and stored in CEF cells. The recombinant virus with the CAGW-HAGB expression framework inserted between nucleotides 112071-112088 of the HVT genome and the ILTV-GDGI expression framework inserted between nucleotides 140079-140730 of the HVT genome was rescued and named H20204. The plaque lesions produced by the above recombinant viruses H20203 and H20204 on CEF are shown in the figure. Figure 2 shown.

[0050] The genomic DNA of recombinant viruses H20203 and H20204 was extracted, and the genomic DNA of the parent virus FC126 was set as a control. The recombinant viruses were identified by PCR using the target gene specific primer LT1348F and the target gene downstream homology arm primer HVT54R or HVT65F (Table 1). The results showed that the recombinant viruses H20203 and H20204 could be amplified to obtain a fragment of about 2687 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. The sequence was correct. The recombinant viral genomic DNA was identified by PCR using the ILTVgI gene-specific primer ILTVgIF and the downstream homology arm primer HVT88R of the target gene GDGI. The result was amplification of a PCR fragment of about 1700 bp ( Figure 3 ), the size was consistent with the expectation. The parent virus FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target genes HAGB and GDGI were correctly inserted into the genome of HVT FC126 strain, and the recombinant viruses H20203 and H20204 were correctly constructed.

[0051] Example 2: In vitro biological characteristics analysis of recombinant turkey herpesvirus expressing H9 subtype AIVHA gene and ILTV gB, gD, gI genes

[0052] 2.1 Detection of recombinant virus expressing H9 subtype AIVHA and ILTV gB, gD, and gI proteins

[0053] The recombinant viruses H20203, H20204 and the parental virus 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 HA, gB, gD, and gI proteins was detected by indirect immunofluorescence assay using HA, gB, gD, and gI polyclonal antibodies as primary antibodies and FITC-labeled goat anti-rabbit IgG as secondary antibodies, respectively. The CEF infected with the parental virus FC126 was used as a negative control. The results showed that the cells infected with the recombinant viruses H20203 and H20204 could react with the HA, gB, gD, and gI polyclonal antibodies, showing green fluorescence signals ( Figure 4 ). No fluorescence was observed in cells infected with the parental HVT virus FC126 strain and in control cells that were not infected. The above results indicate that both recombinant viruses H20203 and H20204 can co-express HA, gB, gD, and gI proteins in infected cells.

[0054] 2.2 Analysis of in vitro replication characteristics of recombinant viruses H20203 and H20204

[0055] The recombinant viruses H20203, H20204 and the parental virus FC126 were inoculated on CEF in a 6-well plate at a dose of 100 plaque-forming units (PFU). The virus-containing cells were collected every 24 hours after infection until 144 hours after infection. The viruses collected at each time point were inoculated into CEF, the number of plaques in the virus solution at each time point was determined, and the in vitro replication kinetic curve was drawn to analyze the in vitro replication characteristics of the recombinant viruses H20203, H20204 and the parental virus FC126 in CEF. The results showed that the replication titers of the recombinant viruses H20203, H20204 and the parental virus FC126 reached the highest peak at 120 hours after infection, which were 1.98×10 5 PFU / ml, 2.11×10 5 PFU / ml and 2.30×10 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 viruses H20203 and H20204 have good replication ability on CEF, and their in vitro replication characteristics are consistent with those of the parent virus FC126 strain.

[0056] 2.3 Genetic stability testing of recombinant viruses H20203 and H20204

[0057] The rescued recombinant viruses H20203 and H20204 were 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 PCR identification of the 20th generation recombinant virus using the target gene HAGB specific primer LT1348F and the target gene downstream homology arm primer HVT54R or HVT65F (Table 1) could amplify a fragment of about 2687bp, which was consistent with the expected size ( Figure 6 ). The recombinant viral genomic DNA was amplified and sequenced using primers CAGF and CAGR, respectively, and it was found that the sequence of the target gene HAGB inserted into the HVT genome was correct. The 20th generation recombinant viral genomic DNA was identified by PCR using the target gene GDGI specific primer ILTVgIF and the target gene downstream homology arm primer HVT88R. The result was amplified to obtain a PCR fragment of about 1700 bp ( Figure 6 ), the size was correct. At the same time, the recombinant viral genomic DNA 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 HVT genome were correct.

[0058] At the same time, the 20th generation recombinant virus was inoculated into CEF cells, and the stability of target gene expression was detected by indirect immunofluorescence test using HA, gB, gD, and gI polyclonal antibodies. The results showed that after the recombinant viruses H20203 and H20204 were continuously propagated on CEF cells to the 20th generation, the exogenous target genes HA, gB, gD, and gI could still be stably expressed ( Figure 7 ). The above results show that the HA, gB, gD, and gI genes inserted into the HVT genome can stably exist during the virus passage process, and the recombinant viruses H20203 and H20204 have good genetic stability.

[0059] Example 3: Safety and immunogenicity testing of recombinant viruses H20203 and H20204

[0060] 3.1 Safety testing of recombinant viruses H20203 and H20204

[0061] The fifth-generation recombinant viruses H20203 and H20204 were inoculated into one-day-old SPF chickens at a dose of 4000 PFU / chicken, and the clinical symptoms of each group of chickens were observed every day after inoculation. 28 days after inoculation, 5 test chickens were randomly selected from each group, and their body weights were weighed to evaluate the effects of the recombinant viruses H20203 and H20204 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 and weighed to observe whether there were symptoms of atrophy or swelling. The results showed that the recombinant viruses H20203 and H20204 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 results of weighing and autopsy showed that the weight of the chickens inoculated with the recombinant viruses H20203 and H20204 was not significantly different from that of the normal uninoculated control group; the experimental 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 viruses H20203 and H20204 are safe for SPF chickens.

[0062] 3.2 Immunoprotective test of recombinant viruses H20203 and H20204 against H9 subtype AIV

[0063] Forty one-day-old SPF chicks were randomly divided into four groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H20203 at a dose of 4000 PFU / chicken, Group 2 was subcutaneously inoculated with the recombinant virus H20204 at a dose of 4000 PFU / chicken, Group 3 was inoculated with the parental virus FC126 at the same dose, and Group 4 was not immunized as a blank control group. 28 days after immunization, Groups 1-3 were nasally challenged with the H9 subtype AIV LC18 strain; Group 4 was not challenged and served as a healthy control group. After the challenge, the chickens were observed for 14 days and the clinical symptoms of the test chickens were recorded. Five days after the challenge, laryngeal and cloacal swabs were collected from each chicken in each group for virus isolation. The collected cotton swabs were repeatedly frozen and thawed three times and then centrifuged. The supernatant was inoculated into 10-day-old SPF chicken embryos through the allantoic cavity. The chicken embryos were placed in an incubator at 37°C for 96 hours, and the allantoic fluid was collected to measure the HA titer. A titer of no less than 1:16 was considered positive for virus isolation. The samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0064] The results showed that after the experimental chickens were challenged with H9 subtype AIV, the virus isolation of 10 chickens in the parental virus 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 recombinant virus H20203 and H20204 inoculation groups of 10 chickens each 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 recombinant viruses H20203 and H20204 against H9 subtype AIV is 100%.

[0065] 3.3 Immunoprotective test of recombinant viruses H20203 and H20204 against ILTV virulent virus

[0066] Forty one-day-old SPF chicks were randomly divided into four groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H20203 at a dose of 4000 PFU / chicken, Group 2 was subcutaneously inoculated with the recombinant virus H20204 at a dose of 4000 PFU / chicken, Group 3 was inoculated with the parental virus FC126 at the same dose, and Group 4 was not immunized as a blank control group. 28 days after immunization, groups 1-3 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 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 H20203 inoculation group were challenged with ILTV, one chicken showed symptoms of 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. All 10 chickens in the recombinant virus H20204 inoculation group survived the ILTV challenge in a healthy manner, without obvious clinical symptoms, and no obvious lesions were found in the autopsy. The above results show that the protection rates of recombinant viruses H20203 and H20204 against ILTV were 90% and 100%, respectively.

Claims

1. A recombinant turkey herpesvirus strain (HVT) that co-expresses the HA gene of H9 subtype avian influenza virus (AIV) 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 the H9 subtype AIVHA gene and the ILTV gB gene and the expression frame ILTV-GDGI containing the ILTVgD and gI genes into the HVT genome.

2. The recombinant turkey herpes virus according to claim 1, characterized in that The expression framework CAGW-HAGB comprising the H9 subtype AIVHA gene and the ILTV gB gene is inserted between nucleotides 95322-95323 or between nucleotides 112071-112088 of the HVT FC126 strain genome, and the GenBank accession number of the HVT FC126 strain genome DNA sequence is AF291866.

3. The recombinant turkey herpes virus according to claim 1, characterized in that The expression framework ILTV-GDGI containing ILTVgD and gI genes is inserted between nucleotides 140079-140730 of the HVT FC126 genome, and the GenBank accession number of the HVT FC126 genome DNA sequence is AF291866.

4. The recombinant turkey herpes virus according to claim 1, characterized in that The expression framework CAGW-HAGB containing the H9 subtype AIVHA gene and ILTVgB gene sequentially contains chicken β-actin promoter, HA gene coding sequence, porcine teschovirus 2A self-cleavage peptide coding sequence, gB gene coding sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit β-globulin polyadenylic acid sequence.

5. The recombinant turkey herpes virus according to claim 1, characterized in that The nucleotide sequence of the expression framework CAGW-HAGB is shown in SEQ ID NO.

4.

6. The recombinant turkey herpes virus according to claim 1, characterized in that The nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.

5.

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 Extract the genomic DNA of HVT FC126 strain, clone the genomic DNA of FC126 strain into pCC1Fos vector, and select 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that clone the genomic DNA fragments of FC126 strain and can be spliced ​​to cover the complete HVT genome; among them, HVT01 contains the nucleotide fragment of 1-30189 of FC126 genome, HVT02 contains the nucleotide fragment of 21572-61476 of FC126 genome, HVT03 contains the nucleotide fragment of 50871-90478 of FC126 genome, HVT04 contains the nucleotide fragment of 73762-108139 of FC126 genome, HVT05 contains the nucleotide fragment of 96776-135815 of FC126 genome, and HVT06 contains the nucleotide fragment of 129602-159160 of FC126 genome; (2) Construction of recombinant cosmids expressing H9 subtype AIVHA gene and ILTV gB gene A recombinant plasmid of an expression framework CAGW-HAGB, which sequentially comprises a chicken β-actin promoter, an HA gene coding sequence, a porcine teschovirus 2A self-cleaving peptide coding sequence, a gB gene coding sequence, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence, is constructed; the expression framework CAGW-HAGB is cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 or the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 by using the Red / ET recombination method, to construct a recombinant cosmid that co-expresses the HA gene and the gB gene, which is named HVT04-53-HAGB or HVT05-65-HAGB; (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 140079-140730 of the HVT genome using the Red / ET recombination method to construct a recombinant cosmid expressing ILTVgD and gI genes, named HVT06-88-GDGI; (4) Rescue of recombinant HVT expressing H9 subtype AIVHA gene and ILTVgB, gD, and gI genes The recombinant cosmids HVT04-53-HAGB, HVT05-65-HAGB, HVT06-88-GDGI and the parental cosmids HVT01, HVT02, HVT03, HVT04, and HVT05 cloned with the HVT FC126 strain genome fragment 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 viruses in which the expression framework CAGW-HAGB was inserted between nucleotides 95322-95323 or 112071-112088 of the HVT FC126 strain genome, and the expression framework ILTV-GDGI was inserted between nucleotides 140079-140730 of the HVT FC126 strain genome were rescued and named H20203 or H20204, respectively.

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.

9. The method according to claim 7, characterized in that The nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.

5.

10. 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 and infectious laryngotracheitis.

Images