Recombinant turkey herpesvirus strain for expressing ILTV gB, gD and gI genes and IBDV classical virulent strain and novel variant VP2 gene and application thereof

Abstract

The invention discloses a recombinant herpesvirus turkey strain for co-expressing ILTVgB, gD and gI genes, an IBDV classical virulent strain and a novel variant VP2 gene and application of the recombinant herpesvirus turkey strain, and belongs to the technical field of medicine or veterinary medicine. The recombinant turkey herpesvirus is prepared by the following steps: inserting an expression frame CAGW-GB2A2 containing an ILTVgB gene and an IBDV novel variant VP2 gene between UL45 and UL46 genes of a turkey herpesvirus genome, and inserting an expression frame CMV-IBDVC2 containing an IBDV classical virulent strain VP2 gene between UL55 and HVT066 genes of the turkey herpesvirus genome. Meanwhile, an expression framework ILTV-GDGI containing ILTVgD and gI genes is inserted into a US2 gene of a turkey herpesvirus genome to obtain the turkey herpesvirus gene. The recombinant turkey herpesvirus strain can provide a good immune protection effect on ILTV virulent strains, IBDV supervirulent strains and novel variants. The invention provides an effective technical means for preventing the chicken infectious laryngotracheitis and the chicken infectious bursal disease.

Description

Technical Field

[0001] The invention relates to a recombinant turkey herpes virus strain which co-expresses the gB, gD and gI genes of infectious laryngotracheitis virus and the VP2 gene of the classical strong strain and the novel variant strain of infectious bursal disease virus of chickens, and the application of the recombinant turkey herpes virus strain in preparing vaccines of infectious laryngotracheitis and infectious bursal disease of chickens, belonging to the technical field of medicine or veterinary medicine. Background Art

[0002] 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.

[0003] Infectious bursal disease (IBD) is an acute, highly contact, 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. In 1957, IBD first broke out in Gambro, Delaware, USA. The pathogen that caused the disease was later called IBDV classical virulent virus (cvIBDV). In 1982, my country first reported the prevalence of IBDV classical virulent virus in Beijing, and then this type of strain was widely prevalent in most poultry-raising areas in my country. Since 2017, atypical IBD outbreaks have occurred in many provinces in my country. The production performance and immune function of the diseased chickens are low, and the bursa of Fabricius is severely atrophied during autopsy. Studies have found that the strain causing the outbreak is a new variant of IBDV (varIBDV). An effective means of preventing and controlling IBD is to implement herd immunization of all chicks. However, the commercial IBD vaccines currently used on the market can no longer provide effective immune protection against varIBDV, which in turn causes severe immunosuppression in infected chickens, becoming a new threat to the healthy development of my country's poultry industry.

[0004] Recombinant live vector vaccines are live vaccines that use genetic engineering technology to construct a virus or bacteria into a vector, and then insert foreign genes into it for expression. Live vector vaccines can express multiple antigens at the same time to make multivalent or multi-linked vaccines, which can prevent multiple diseases with one shot. It is one of the main directions of vaccine research and development today and in the future. Herpesvirus of turkeys (HVT) is a herpes virus that is non-pathogenic to chickens and is widely used in the prevention of Marek's disease in chickens at home and abroad. As a herpes virus, HVT has a large genome and many replication-non-essential genes that can be inserted or replaced by foreign genes. It is an ideal viral vector for constructing recombinant live vector vaccines. Compared with other viral vectors, HVT has many advantages as a viral vector. It is non-pathogenic to chickens and other animals and is safe to use; the immune effect of HVT vaccine is not interfered by maternal antibodies and can be used for early immunization of one-day-old chicks in hatcheries; after vaccination, the virus exists in the chicken for a long time, stimulating the body to produce high antibody levels and maintain them for life, and lifelong immunity can be obtained with one vaccination; HVT vaccine not only has low production costs, but also can be freeze-dried, easy to store and transport. Summary of the invention

[0005] The present invention aims to provide a recombinant turkey herpes virus strain which co-expresses the gB, gD and gI genes of infectious laryngotracheitis virus and the VP2 gene of the classical virulent strain and the novel variant strain of infectious bursal disease virus of chickens, and its application in the preparation of infectious laryngotracheitis and infectious bursal disease vaccines of chickens.

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

[0007] The invention discloses a recombinant turkey herpesvirus strain for co-expressing infectious laryngotracheitis virus gB, gD and gI genes and VP2 genes of classical virulent strains and novel variant strains of infectious bursal disease virus of chicken. The recombinant turkey herpesvirus strain is obtained by inserting an expression frame CAGW-GB2A2 containing ILTVgB gene and VP2 gene of novel variant strain of IBDV between UL45 and UL46 genes of turkey herpesvirus genome, inserting an expression frame CMV-IBDVC2 containing VP2 gene of classical virulent strain of IBDV between UL55 and HVT066 genes of turkey herpesvirus genome, and simultaneously inserting an expression frame ILTV-GDGI containing ILTVgD and gI genes into US2 gene of turkey herpesvirus genome.

[0008] Among them, preferably, the expression framework CAGW-GB2A2 contains chicken β-actin promoter, ILTV gB gene and IBDV new variant VP2 gene coding sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit β-globulin polyadenylation sequence, wherein the gB gene and IBDA2 gene coding sequence are connected by the porcine teschovirus 2A coding sequence.

[0009] Among them, preferably, the nucleotide sequence of the expression framework CAGW-GB2A2 is shown in SEQ ID NO.4.

[0010] Among them, preferably, the expression framework CMV-IBDVC2 contains a mouse cytomegalovirus promoter, an IBDV classic virulent strain VP2 gene coding sequence and an SV40 polyadenylation sequence.

[0011] Among them, preferably, the nucleotide sequence of the expression frame CMV-IBDVC2 is shown as SEQ ID NO.6.

[0012] Preferably, the nucleotide sequence of the expression framework ILTV-GDGI is as shown in SEQ ID NO.7.

[0013] Preferably, the turkey herpes virus is the turkey herpes virus FC126 strain, and the GenBank accession number of the genomic DNA sequence of the turkey herpes virus FC126 strain is AF291866.

[0014] Furthermore, the present invention also proposes a method for constructing the recombinant turkey herpes virus strain, which comprises the steps of co-transfecting CEF cells with recombinant cosmids H434, H481, and H159 containing genomic DNA fragments of turkey herpes virus, recombinant cosmid H483-53-GB2A2 containing ILTVgB gene and IBDV novel variant VP2 gene expression framework CAGW-GB2A2, recombinant cosmid H361-65-IBDVC2 containing IBDV classical virulent strain VP2 gene expression framework CMV-IBDVC2, and recombinant cosmid H220-88-GDGI containing ILTV gD and gI gene expression framework ILTV-GDGI, and then obtaining the recombinant turkey herpes virus strain by virus rescue;

[0015] The recombinant turkey herpes virus strain is the turkey herpes virus FC126 strain, and the GenBank accession number of the genomic DNA sequence is AF291866;

[0016] The recombinant cosmid H434 comprises a nucleotide fragment of positions 1 to 40394 of the genome of the turkey herpes virus FC126 strain, the recombinant cosmid H481 comprises a nucleotide fragment of positions 31586 to 69191 of the genome of the turkey herpes virus FC126 strain, and the recombinant cosmid H159 comprises a nucleotide fragment of positions 58293 to 92645 of the genome of the turkey herpes virus strain FC126;

[0017] The recombinant cosmid H483-53-GB2A2 is obtained by inserting the expression frame CAGW-GB2A2 between the UL45 and UL46 genes of the FC126 strain genome of the turkey herpes virus on the basis of the recombinant cosmid H483, and the recombinant cosmid H483 contains the nucleotide fragment of the 76940-110712 position of the FC126 strain genome of the turkey herpes virus;

[0018] The recombinant cosmid H361-65-IBDVC2 is obtained by inserting the expression frame CMV-IBDVC2 between the UL55 and HVT066 genes of the FC126 strain of turkey herpesvirus on the basis of the recombinant cosmid H361, and the recombinant cosmid H361 contains the nucleotide fragment of the 103006-134393 position of the genome of the FC126 strain of turkey herpesvirus;

[0019] The recombinant cosmid H220-88-GDGI is obtained by inserting the expression frame ILTV-GDGI into the US2 gene of the FC126 strain genome of turkey herpesvirus on the basis of the recombinant cosmid H220, and the recombinant cosmid H220 contains the nucleotide fragment of positions 123621-159160 of the FC126 strain genome of turkey herpesvirus;

[0020] Among them, preferably, the nucleotide sequence of the expression framework CAGW-GB2A2 is shown as SEQ ID NO.4; the nucleotide sequence of the expression framework CMV-IBDVC2 is shown as SEQ ID NO.6; and the nucleotide sequence of the expression framework ILTV-GDGI is shown as SEQ ID NO.7.

[0021] Furthermore, the present invention also proposes the use of the recombinant turkey herpesvirus strain in the preparation of vaccines for preventing infectious laryngotracheitis and infectious bursal disease.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention provides a recombinant turkey herpesvirus strain that co-expresses the gB, gD and gI genes of infectious laryngotracheitis virus and the VP2 genes of the classical strong strain and the novel variant of infectious bursal disease virus of chickens. The recombinant turkey herpesvirus strain can co-express the ILTV gB, gD, gI proteins and the IBDV VP2 protein in infected cells, has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus HVT FC126 strain, and has good genetic stability. The recombinant virus strain is safe for SPF chickens, has a good immune protection effect on the strong ILTV virus, and can also provide a good immune protection effect on the super strong strain and the novel variant of IBDV. Therefore, the recombinant virus strain can be used to prepare a vaccine for preventing infectious laryngotracheitis and infectious bursal disease of chickens. The invention provides an effective technical means for preventing infectious laryngotracheitis and infectious bursal disease of chickens. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The PCR identification results of the recombinant cosmids H483-53-GB2A2, H361-65-IBDVC2 and H220-88-GDGI;

[0025] Figure 2 This is the plaque lesion produced by the recombinant virus H20702 on CEF;

[0026] Figure 3 This is the PCR identification result of the recombinant virus H20702 genomic DNA;

[0027] Figure 4 The results of the expression test of ILTV gB, gD, gI and IBDVVP2 proteins in CEF infected with the recombinant virus H20702 are shown;

[0028] Figure 5 is the replication kinetic curve of the recombinant virus H20702 on CEF cells;

[0029] Figure 6 This is the PCR test result of the genetic stability of the recombinant virus H20702;

[0030] Figure 7 The results of indirect immunofluorescence test of the 20th generation recombinant virus H20702 expressing ILTV gB, gD, gI and IBDVVP2 proteins;

[0031] Figure 8 This is the survival curve of chickens immunized with the recombinant virus H20702 after being challenged with a very virulent strain of IBDV;

[0032] Fig. 9 The statistical results of the bursal index of chickens immunized with the recombinant virus H20702 after being challenged with the new variant strain of IBDV. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1: Construction and identification of recombinant turkey herpesviruses co-expressing ILTV gB, gD, gI genes and IBDV classic virulent strain and novel variant VP2 gene

[0035] 1.1 Establishment of the multi-fragment cosmid rescue system of HVTFC126 strain

[0036] 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 in segments, and the constructed recombinant cosmids were terminally sequenced with primers pCC1F and pCC1R (see Table 1 for details). According to the sequencing results, 6 recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned HVT FC126 strain genomic DNA fragments and could be spliced ​​to cover the complete HVT FC126 strain 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. The appearance of cytopathic effects can be observed 4-5 days after transfection, indicating that the parental virus HVTFC126 strain was rescued.

[0037] 1.2 Construction and identification of recombinant cosmids expressing ILTVgB gene and VP2 gene of novel IBDV variant

[0038] According to the genomic sequence of ILTVWG strain (GenBank accession number JX458823), the ILTV gB gene was synthesized after chicken codon optimization design, and its nucleotide sequence is shown in SEQ ID NO.1. According to the VP2 gene sequence of varIBDV FJ-18 strain (GenBank accession number OK167034), the porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence was added to its 5' end, and the target gene IBDA2 was synthesized after chicken codon optimization design, and its nucleotide sequence is shown in SEQ ID NO.2. The target gene GB2A2 containing the ILTVgB gene and IBDA2 gene coding sequences was obtained by fusion PCR amplification using the primers LTGBEF, LTGB2AP1R, LTGB2AP2F and avVP2WCR in Table 1, and its nucleotide sequence is shown in SEQ ID No.3, wherein the gB gene and IBDA2 gene coding regions are connected by the self-cleavage peptide P2A coding sequence. The PCR product was cloned into the downstream of the chicken β-actin promoter of the pCAGGS vector through the EcoRI and ClaI restriction sites, and the woodchuck hepatitis virus post-transcriptional regulatory sequence (GenBank accession number MQ208857.1) was inserted between the target gene GB2A2 and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-GB2A2 that co-expressed the ILTV gB gene and the IBDV new variant VP2 gene. The recombinant plasmid was sequenced using the primers CAGF and CAGR in Table 1, and it was found that the cloned target gene expression cassette sequence was correct.

[0039] According to the instructions of the Counter Selection BAC Modification Kit, the expression framework CAGW-GB2A2 (shown in SEQ ID No. 4) containing the chicken β-actin promoter, the target gene GB2A2, the woodchuck hepatitis virus post-transcriptional regulatory sequence and the rabbit β-globulin polyadenylation sequence was cloned into the recombinant cosmid H483 between the UL45 and UL46 genes of the HVT genome, and the recombinant cosmid H483-53-GB2A2 expressing the ILTV gB gene and the varIBDVVP2 gene was constructed. The target gene primer LTGB2AP2F in Table 1 and the target gene downstream homology arm primer HVT54R were used to identify whether the recombinant cosmid H483-53-GB2A2 was inserted into the target gene GB2A2 expression framework, and the PCR product was sequenced and identified. The results showed that the PCR fragment (about 2800 bp in size) was obtained. Figure 1 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the IBDA2 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 H483-53-GB2A2 was constructed correctly.

[0040] 1.3 Construction and identification of recombinant cosmids expressing the VP2 gene of classic IBDV virulent strains

[0041] According to the cvIBDVFaragher 52 / 70 strain VP2 gene sequence (GenBank accession number HG974565), the target gene IBDVC2 was synthesized, and its nucleotide sequence was shown in SEQ ID NO.5. The target gene IBDVC2 was cloned into the pUC57 vector to obtain the recombinant plasmid pUC57-IBDVC2. The recombinant plasmid pUC57-IBDVC2 cloned with the IBDVC2 gene was used as a template, and the IBDVC2 gene was obtained by PCR amplification using the primers IBDVC2F and IBDVC2R in Table 1. The purified PCR product was cloned into the pCMV vector between the mouse cytomegalovirus promoter and the SV40 polyadenylation sequence to construct the recombinant plasmid pCMV-IBDVC2 expressing the cvIBDVVP2 gene.

[0042] According to the instructions of the Counter Selection BAC Modification Kit, the expression framework CMV-IBDVC2 (shown in SEQ ID NO.6) containing the mouse cytomegalovirus promoter, the VP2 gene of the classical strong strain of IBDV, and the SV40 polyadenylation sequence was cloned into the recombinant cosmid H361 between the UL55 and HVT066 genes of the HVT genome to construct the recombinant cosmid H361-65-IBDVC2 expressing the cvIBDVVP2 gene. The target gene primer IBDVC2F in Table 1 and the downstream homology arm primer HVT65F of the target gene were used to identify whether the recombinant cosmid H361-65-IBDVC2 was inserted into the target gene IBDVC2 expression framework, and the PCR product was sequenced and identified. The results showed that the PCR fragment (about 1800 bp in size) was obtained. Figure 1 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the IBDVC2 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.

[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 was amplified by PCR, and its nucleotide sequence is shown in SEQ ID NO.7. According to the instructions of Counter Selection BACModificationKit kit, the above expression framework ILTV-GDGI was cloned into the US2 gene of the HVT genome in the recombinant cosmid H220 using the Red / ET recombination method to construct the recombinant cosmid H220-88-GDGI expressing ILTV gD and gI genes. The target gene primer ILTVgIF in Table 1 and the target gene downstream homology arm primer HVTUS2R were used to identify whether the gD and gI gene expression frameworks were inserted into the recombinant cosmid H220-88-GDGI by PCR, and the PCR products were sequenced and identified. The results showed that the PCR fragment with a size of about 1900 bp ( Figure 1 ), the size was consistent with expectations. The recombinant cosmid was PCR amplified and sequenced using primers HVTUS2F and HVTUS2R in Table 1. 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 H220-88-GDGI was constructed correctly.

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

[0046]

[0047] 1.5 Rescue and identification of recombinant HVT co-expressing ILTV gB, gD, gI genes and IBDV classic virulent strain and novel variant VP2 gene

[0048] The recombinant cosmids H483-53-GB2A2, H361-65-IBDVC2, H220-88-GDGI and other parental cosmids H434, H481, and H159 cloned with HVT FC126 strain genomic fragments were extracted using a plasmid extraction kit. The above six recombinant cosmids were co-transfected into CEF cells by calcium phosphate transfection, and the viruses were harvested after plaque lesions appeared after 4-5 days of culture. The harvested virus was serially passaged and stored in CEF cells, and a recombinant virus was rescued in which the ILTV gB gene and varIBDVVP2 gene expression framework were inserted between the UL45 and UL46 genes of the HVT FC126 strain genome, the cvIBDVVP2 gene expression framework was inserted between the UL55 and HVT066 genes of the HVT FC126 strain genome, and the ILTV gD and gI gene expression frameworks were inserted into the US2 gene of the HVT FC126 strain genome. The recombinant virus was named H20702. The plaque lesions produced by the recombinant virus on CEF were as shown in FIG. Figure 2 shown.

[0049] The recombinant virus genomic DNA was extracted, and the parent virus HVT FC126 strain genomic DNA was used as a control. The recombinant virus genomic DNA was identified by PCR using the varIBDVVP2 gene specific primer LTGB2AP2F and the target gene GB2A2 downstream homology arm primer HVT54R. The result was amplification of a PCR fragment of about 2800 bp ( Figure 3 ), the size was consistent with expectations. The recombinant virus genomic DNA was identified by PCR using the cvIBDVVP2 gene-specific primer IBDVC2F and the target gene IBDVC2 downstream homology arm primer HVT65F. The result was amplification of a PCR fragment of about 1800 bp ( Figure 3 ), the size was consistent with expectations. In addition, the recombinant viral genomic DNA was identified by PCR using the ILTVgI gene-specific primer ILTVgIF and the target gene GDGI downstream homology arm primer HVTUS2R, and the result was amplified to obtain a PCR fragment of approximately 1900 bp ( Figure 3 ), the size was consistent with the expectation. However, the parent virus HVT FC126 strain had no such target gene insertion, and the PCR amplification results were all negative. The above results showed that the target genes GB2A2, IBDVC2 and GDGI were correctly inserted into the genome of HVT FC126 strain, and the recombinant virus was correctly constructed.

[0050] Example 2: Analysis of in vitro biological characteristics of recombinant turkey herpesvirus H20702 co-expressing ILTV gB, gD, gI genes and IBDV classic virulent strain and novel variant VP2 gene

[0051] 2.1 Detection of recombinant virus H20702 expressing ILTVgB, gD, gI and IBDVVP2 proteins

[0052] The recombinant virus H20702 and the parental virus HVT FC126 strain were inoculated into CEF cells cultured in 6-well plates, with each virus strain inoculated into 4 wells, and cells without virus infection 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 ILTV gB, gD, gI polyclonal antibodies and IBDV VP2 monoclonal antibodies were used as primary antibodies, and FITC-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies. Indirect immunofluorescence assay was used to detect the expression of ILTV gB, gD, gI and IBDVVP2 proteins, and CEF infected with the parental virus HVT FC126 strain was used as a negative control. The results showed that cells infected with the recombinant virus H20702 could react with ILTV gB, gD, gI polyclonal antibodies and IBDVVP2 monoclonal antibodies, showing green fluorescence signals ( Figure 4 ). No fluorescence was observed in cells infected with the parent virus HVT FC126 strain and in control cells that were not infected. The above results indicate that the recombinant virus H20702 can co-express ILTV gB, gD, gI proteins and IBDV VP2 protein in infected cells.

[0053] 2.2 Analysis of in vitro replication characteristics of recombinant virus H20702

[0054] The recombinant virus H20702 and the parent 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 virus H20702 and the parent virus FC126 strain in CEF. The results showed that the replication titers of the recombinant virus H20702 and the parent virus FC126 strain reached the highest peak 120 hours after infection, which were 1.94×10 5 PFU / ml and 1.85×10 5 PFU / ml, the titer of the recombinant virus at each time point after infection with CEF was not significantly different from that of the parent virus FC126 strain (P>0.05)( Figure 5 ). The above results show that the recombinant virus H20702 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus FC126 strain.

[0055] 2.3 Genetic stability test of recombinant virus H20702

[0056] The rescued recombinant virus H20702 was continuously passaged on CEF to the 20th generation, and the genomic DNA of the 20th generation recombinant virus was extracted for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the recombinant virus genome. The results showed that the PCR identification of the 20th generation recombinant virus using the target gene GB2A2 specific primer LTGB2AP2F and the target gene downstream homology arm primer HVT54R in Table 1 can amplify a fragment of about 2800 bp, which is consistent with the expected size ( Figure 6 ). The recombinant virus genomic DNA was amplified and sequenced using primers CAGF and CAGR in Table 1, and it was found that the sequence of the target gene GB2A2 inserted into the HVT genome was correct. The 20th generation recombinant virus was identified by PCR using the target gene IBDVC2 specific primer IBDVC2F and the target gene downstream homology arm primer HVT65F in Table 1. A fragment of about 1800 bp was amplified, which was consistent with the expected size ( Figure 6 ). The sequencing results showed that the PCR product contained the cvIBDVVP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The target gene GDGI specific primer ILTVgIF and the target gene downstream homology arm primer HVTUS2R in Table 1 were used to perform PCR identification on the 20th generation recombinant virus genomic DNA, and the result was amplified to obtain a PCR fragment of about 1900 bp ( Figure 6 ), the size was correct. At the same time, the recombinant viral genomic DNA was amplified by PCR and sequenced using primers HVTUS2F and HVTUS2R, and the results showed that the ILTVgD and gI gene expression framework sequences inserted into the genome of the HVT FC126 strain were correct.

[0057] 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 ILTVgB, gD, gI polyclonal antibodies and IBDVVP2 monoclonal antibodies. The results showed that after the recombinant virus H20702 was continuously propagated on CEF cells to the 20th generation, the exogenous target genes gB, gD, gI and VP2 could still be stably expressed ( Figure 7 ). The above results show that the ILTVgB, gD, gI and IBDVVP2 genes inserted into the HVT genome can stably exist during the virus passage process, and the recombinant virus H20702 has good genetic stability.

[0058] Example 3: Safety and immunogenicity testing of recombinant virus H20702

[0059] 3.1 Safety testing of recombinant virus H20702

[0060] The fifth-generation recombinant virus H20702 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 virus H20702 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the organs such as the bursa of Fabricius, thymus, spleen, and liver were collected, weighed, and observed for atrophy or swelling. The results showed that the recombinant virus H20702 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 virus H20702 inoculation group was no significantly different from that of the normal uninoculated control group; the test chickens were autopsied, and the bursa of Fabricius, thymus, spleen, liver and other organs were collected. The results showed that the above organs were normal and no obvious clinical lesions were found. The above results show that the recombinant virus H20702 is safe for SPF chickens.

[0061] 3.2 Immunoprotection test of recombinant virus H20702 against ILTV virulent virus

[0062] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H20702 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 and 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 FC126 strain inoculation group was challenged with the virulent ILTV, all 10 test chickens showed symptoms such as lethargy, ruffled 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. All 10 chickens in the recombinant virus H20702 inoculation group survived the ILTV virulent infection without obvious clinical symptoms and no obvious lesions were found in the autopsy. The above results show that the recombinant virus H20702 has a good immune protection effect against the ILTV virulent infection.

[0063] 3.3 Immunoprotective test of recombinant virus H20702 against IBDV super-virulent strains and new variants

[0064] 60 one-day-old SPF chicks were randomly divided into 3 groups, 20 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H20702 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, 10 chickens from Group 1 and Group 2 were respectively challenged with the IBDV super-virulent HN strain, 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 remaining 10 chickens in Group 1 and Group 2 were challenged with the new variant strain FJ of IBDV, 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 lesions and the bursa weight ratio (F / B) and bursa index (BBIX) were counted. F / B=(bursa weight / body weight)×1000, BBIX=bursa weight ratio of chickens in the experimental group / cyst ratio of chickens in the blank control group; when BBIX is less than 0.7, it is judged as bursa atrophy.

[0065] The results showed that after the parent virus HVT FC126 strain was challenged with IBDV super-virulent virus, all 10 chickens showed symptoms such as lethargy, ruffled feathers, and lying on the ground. Eight of the infected chickens died ( Figure 8 ), autopsy showed that the bursa of Fabricius of all 10 chickens showed atrophy, strip-shaped bleeding or yellow jelly-like exudation lesions; all 10 chickens in the recombinant virus H20702 inoculation group survived after being challenged with IBDV super-strong virus, and autopsy found no obvious lesions in the bursa of Fabricius. No deaths occurred in the parent virus FC126 inoculation group after being challenged with the new variant of IBDV, but the bursa of all 10 chickens showed atrophy lesions, and BBIX was less than 0.7 ( Fig. 9 ); All the rats in the recombinant virus H20702 immunization group survived the challenge with the new IBDV variant strain. The autopsy revealed no obvious lesions in the bursa of Fabricius, and the BBIX was greater than 0.7. The above results show that the recombinant virus H20702 can provide good immune protection against both the super-virulent strains and the new variants of IBDV.

Claims

1. A recombinant turkey herpesvirus strain (HVT) that co-expresses gB, gD and gI genes of infectious laryngotracheitis virus (ILTV) and VP2 genes of classical virulent strains and novel variants of infectious bursal disease virus (IBDV), characterized in that: The recombinant turkey herpes virus is obtained by inserting an expression frame CAGW-GB2A2 containing an ILTV gB gene and an IBDV novel variant VP2 gene between UL45 and UL46 genes of a turkey herpes virus genome, inserting an expression frame CMV-IBDVC2 containing an IBDV classical strong strain VP2 gene between UL55 and HVT066 genes of a turkey herpes virus genome, and inserting an expression frame ILTV-GDGI containing ILTV gD and gI genes into a US2 gene of a turkey herpes virus genome.

2. The recombinant turkey herpes virus strain according to claim 1, characterized in that The expression framework CAGW-GB2A2 comprises chicken β-actin promoter, ILTVgB gene and IBDV novel variant VP2 gene coding sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit β-globulin polyadenylation sequence, wherein the gB gene and VP2 gene coding sequence are connected by porcine Teschovirus 2A coding sequence.

3. The recombinant turkey herpes virus strain according to claim 2, characterized in that The nucleotide sequence of the expression framework CAGW-GB2A2 is shown in SEQ ID NO.

4.

4. The recombinant turkey herpes virus strain according to claim 1, characterized in that The expression framework CMV-IBDVC2 comprises a mouse cytomegalovirus promoter, an IBDV classic strong strain VP2 gene coding sequence and an SV40 polyadenylation sequence.

5. The recombinant turkey herpes virus strain according to claim 4, characterized in that The nucleotide sequence of the expression framework CMV-IBDVC2 is shown in SEQ ID NO.

6.

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

7.

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

8. A method for constructing the recombinant turkey herpes virus strain according to any one of claims 1 to 7, characterized in that: The method comprises the steps of co-transfecting CEF cells with recombinant cosmids H434, H481, H159 containing genomic DNA fragments of turkey herpesvirus, recombinant cosmid H483-53-GB2A2 containing ILTV gB gene and IBDV novel variant VP2 gene expression framework CAGW-GB2A2, recombinant cosmid H361-65-IBDVC2 containing IBDV classical virulent strain VP2 gene expression framework CMV-IBDVC2, and recombinant cosmid H220-88-GDGI containing ILTV gD and gI gene expression framework ILTV-GDGI, and then obtaining the recombinant turkey herpesvirus strain by virus rescue; The recombinant turkey herpes virus strain is the turkey herpes virus FC126 strain, and the GenBank accession number of the genomic DNA sequence is AF291866; The recombinant cosmid H434 comprises a nucleotide fragment of positions 1 to 40394 of the genome of the turkey herpes virus FC126 strain, the recombinant cosmid H481 comprises a nucleotide fragment of positions 31586 to 69191 of the genome of the turkey herpes virus FC126 strain, and the recombinant cosmid H159 comprises a nucleotide fragment of positions 58293 to 92645 of the genome of the turkey herpes virus FC126 strain; The recombinant cosmid H483-53-GB2A2 is obtained by inserting the expression frame CAGW-GB2A2 between the UL45 and UL46 genes of the FC126 strain genome of the turkey herpes virus on the basis of the recombinant cosmid H483, and the recombinant cosmid H483 contains the nucleotide fragment of the 76940-110712 position of the FC126 strain genome of the turkey herpes virus; The recombinant cosmid H361-65-IBDVC2 is obtained by inserting the expression frame CMV-IBDVC2 between the UL55 and HVT066 genes of the FC126 strain of turkey herpesvirus on the basis of the recombinant cosmid H361, and the recombinant cosmid H361 contains the nucleotide fragment of the 103006-134393 position of the genome of the FC126 strain of turkey herpesvirus; Among them, the recombinant cosmid H220-88-GDGI is obtained by inserting the expression frame ILTV-GDGI into the US2 gene of the FC126 strain genome of the turkey herpesvirus on the basis of the recombinant cosmid H220, and the recombinant cosmid H220 contains the nucleotide fragment of positions 123621-159160 of the FC126 strain genome of the turkey herpesvirus.

9. The method according to claim 8, characterized in that The nucleotide sequence of the expression framework CAGW-GB2A2 is shown in SEQ ID NO.4; the nucleotide sequence of the expression framework CMV-IBDVC2 is shown in SEQ ID NO.6; and the nucleotide sequence of the expression framework ILTV-GDGI is shown in SEQ ID NO.

7.

10. Use of the recombinant turkey herpesvirus strain according to any one of claims 1 to 7 in the preparation of a vaccine for preventing infectious laryngotracheitis and infectious bursal disease.

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