Recombinant herpesvirus turkey for co-expressing CIAV VP1 and VP2 genes and IBDV VP2 gene as well as construction method and application of recombinant herpesvirus turkey

Abstract

The invention discloses a recombinant herpesvirus turkey for co-expressing chicken infectious anemia virus VP1 and VP2 genes and an infectious bursal disease virus VP2 gene as well as a construction method and application of the recombinant herpesvirus turkey, and belongs to the technical field of medicine or veterinary medicine. According to the invention, by using a recombinant cloning technology, chicken infectious anemia virus VP1 and VP2 gene expression cassettes are inserted between 95322-95323 nucleotides of a turkey herpesvirus FC126 strain, and meanwhile, an infectious bursal disease virus VP2 gene expression cassette is inserted between 112071-112088 nucleotides of a genome of the turkey herpesvirus FC126 strain; and rescuing to obtain the recombinant turkey herpesvirus vaccine strain for co-expressing the chicken infectious anemia virus VP1 and VP2 genes and the infectious bursal disease virus VP2 gene. The invention provides a more convenient and effective technical means for simultaneously preventing chicken infectious anemia and chicken infectious bursal disease, and can achieve the effect of preventing multiple diseases by one injection.

Description

Technical Field

[0001] The invention relates to a recombinant turkey herpes virus which co-expresses chicken infectious anemia virus VP1 and VP2 genes and chicken infectious bursal disease virus VP2 gene, and a construction method and application thereof, and belongs to the field of genetic engineering vaccines. Background Art

[0002] Chicken Infectious Anemia (CIA) is an immunosuppressive disease caused by Chicken Infectious Anemia Virus (CIAV), characterized by aplastic anemia and systemic lymphoid tissue atrophy in chicks. In 1992, the virus was first isolated in my country. Subsequently, through CIA serological surveys and virus isolation and identification of chickens in many regions of the country, it was found that the disease was common in many chicken farms in my country, and the positive rate in some chicken farms was as high as 40% to 70%. The morbidity rate of chicks infected with CIAV is generally 20% to 60%, and can reach 100% in high cases. The mortality rate is generally 5% to 10%, and can reach more than 60% in severe cases. The disease mainly causes aplastic anemia and immunosuppression in chicks, increases the susceptibility of chickens to other pathogens and reduces the immune response to vaccines, making secondary infections and vaccine immune failures more likely to occur, causing huge economic losses to the breeding industry. The VP1 protein encoded by CIAV is the only capsid protein and main immunogenic protein of the virus; VP2 is a non-structural protein encoded by the virus, which enables VP1 to form the correct conformation during the viral assembly process and assists VP1 in producing neutralizing antibodies.

[0003] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive infectious disease caused by infectious bursal disease virus (IBDV) that mainly harms chicks. The IBDV genome consists of two double-stranded RNA segments. The VP2 protein encoded by segment A is the only capsid protein of IBDV, and is also the main virulence gene and host protective antigen of IBDV. In 1957, IBD first broke out in Gambro, Delaware, USA. The pathogen that caused the disease was later called the classic IBDV virulent virus. 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 that caused the outbreak is a new variant of IBDV (varIBDV). An effective means of preventing and controlling IBD is to implement herd immunization for all chicks. However, varIBDV can break through the immune protection of the commercial IBD vaccines currently used on the market, thereby causing severe immunosuppression in the infected chickens, becoming a new threat to the healthy development of my country's poultry industry.

[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. 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 recombinant turkey herpes virus capable of simultaneously preventing chicken infectious anemia and chicken infectious bursal disease, and a construction method and application thereof.

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

[0007] First, the present invention proposes a recombinant turkey herpesvirus capable of co-expressing the VP1 and VP2 genes of chicken infectious anemia virus and the VP2 gene of chicken infectious bursal disease virus. The recombinant turkey herpesvirus is obtained by inserting an expression frame CAGW-CAVP12 comprising a chicken β-actin promoter, CIAV VP1 and VP2 gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylic acid sequence between nucleotides 95322-95323 of the HVT genome, and simultaneously inserting an expression frame CMV-IBDVA2 comprising a mouse cytomegalovirus promoter, an IBDV VP2 gene coding sequence, and an SV40 polyadenylic acid sequence between nucleotides 112071-112088 of the HVT genome.

[0008] Among them, preferably, the CIAV VP1 and VP2 genes are obtained by connecting the VP1 and VP2 gene coding regions with the porcine teschovirus 2A self-cleavage peptide coding sequence, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0009] Among them, preferably, the expression frame CAGW-CAVP12 comprises in sequence: chicken β-actin promoter, CIAVVP1 and VP2 gene coding sequences, woodchuck hepatitis virus post-transcriptional regulatory sequences and rabbit β-globulin polyadenylic acid sequences, and the nucleotide sequence of the expression frame CAGW-CAVP12 is shown in SEQ ID NO.2.

[0010] Among them, preferably, the IBDV VP2 gene coding sequence is synthesized based on the VP2 gene sequence of the varIBDVFJ-18 strain with GenBank accession number OK167034 after chicken codon optimization design, and its nucleotide sequence is shown in SEQ ID NO.3.

[0011] Among them, preferably, the IBDV is a novel variant of infectious bursal disease virus (varIBDV).

[0012] Among them, preferably, the nucleotide sequence of the expression framework CMV-IBDVA2 is shown in SEQ ID NO.4.

[0013] Among them, preferably, the HVT is HVTFC126 strain, and the GenBank accession number of the genome sequence is AF291866.

[0014] Secondly, the present invention also proposes a method for recombining the turkey herpes virus strain, comprising the following steps:

[0015] (1) Establishment of the multi-fragment cosmid rescue system of HVT strain

[0016] 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 HVT genome, were selected; among them, HVT01 contained the nucleotide fragment of 1-30189 of the HVT FC126 genome, HVT02 contained the nucleotide fragment of 21572-61476 of the HVT FC126 genome, HVT03 contained the nucleotide fragment of 50871-90478 of the HVTFC126 genome, HVT04 contained the nucleotide fragment of 73762-108139 of the HVT FC126 genome, and HVT05 contained the nucleotide fragment of 1-30189 of the HVT FC126 genome. The nucleotide fragment of FC126 genome is from 96776 to 135815, and HVT06 contains the nucleotide fragment of HVTFC126 genome from 129602 to 159160;

[0017] (2) Construction of recombinant cosmids expressing CIAV VP1 and VP2 genes

[0018] A recombinant expression plasmid comprising a chicken β-actin promoter, CIAV VP1 and VP2 gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence expression framework CAGW-CAVP12 was constructed, wherein the VP1 and VP2 gene coding regions were connected by a porcine Teschovirus 2A self-cleaving peptide coding sequence; the expression framework CAGW-CAVP12 was cloned into the HVT FC126 genome in the recombinant cosmid HVT04 between nucleotides 95322 and 95323 by the Red / ET recombination method, and a recombinant cosmid that co-expressed CIAV VP1 and VP2 genes was constructed and named HVT04-53-CAVP12;

[0019] (3) Construction of recombinant cosmid expressing IBDV VP2 gene

[0020] A recombinant expression plasmid comprising a mouse cytomegalovirus promoter, a varIBDV VP2 gene coding sequence and an SV40 polyadenylation sequence expression frame CMV-IBDVA2 was constructed; the expression frame CMV-IBDVA2 was cloned into the recombinant cosmid HVT05 between nucleotides 112071 and 112088 of the HVT FC126 genome using the Red / ET recombination method to construct a recombinant cosmid expressing the IBDV VP2 gene, named HVT05-65-IBDVA2;

[0021] (3) Rescue of recombinant HVT co-expressing CIAV VP1 and VP2 genes and IBDV VP2 gene

[0022] The recombinant cosmids HVT04-53-CAVP12, HVT05-65-IBDVA2 and the other four parental cosmids HVT01, HVT02, HVT03 and HVT06 cloned with the genomic fragments of HVT FC126 strain were extracted; 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. The recombinant virus in which the expression framework CAGW-CAVP12 was inserted between nucleotides 95322-95323 of the HVT FC126 strain genome and the expression framework CMV-IBDVA2 was inserted between nucleotides 112071-112088 of the HVT FC126 strain genome was rescued and named H21001.

[0023] Finally, the present invention also proposes the use of the recombinant turkey herpes virus strain in the preparation of a vaccine for simultaneously preventing chicken infectious anemia and chicken infectious bursal disease.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention utilizes the recombinant cloning technology to insert the chicken infectious anemia virus VP1 and VP2 gene expression frameworks into the 95322-95323 nucleotides of the turkey herpes virus FC126 strain, and simultaneously insert the infectious bursal disease virus VP2 gene expression framework into the 112071-112088 nucleotides of the turkey herpes virus FC126 strain genome, thereby rescuing and obtaining the recombinant turkey herpes virus vaccine strain H21001 that co-expresses the chicken infectious anemia virus VP1 and VP2 genes and the infectious bursal disease virus VP2 gene. The recombinant virus H21001 has good replication ability and genetic stability on CEF, and has good immune protection effect on both the strong toxin of CIAV and the strong toxin of the new variant strain of IBDV.

[0026] In summary, the invention provides a more convenient and effective technical means for preventing both infectious anemia and infectious bursal disease of chickens, and can achieve the effect of preventing multiple diseases with one injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The PCR identification results of the recombinant cosmids HVT04-53-CAVP12 and HVT05-65-IBDVA2;

[0028] Figure 2 This is the plaque lesion produced by the recombinant virus H21001 on CEF;

[0029] Figure 3 This is the PCR identification result of the recombinant virus H21001 genomic DNA;

[0030] Figure 4 Indirect immunofluorescence assay was used to detect the expression of CIAV VP1 and VP2 proteins and IBDV VP2 protein in H21001-infected CEFs;

[0031] Figure 5 is the replication kinetic curve of the recombinant virus H21001 on CEF cells;

[0032] Figure 6 This is the PCR test result of the genetic stability of the recombinant virus H21001;

[0033] Figure 7 The results of indirect immunofluorescence test of the 20th generation recombinant virus H21001 expressing CIAV VP1, VP2 protein and IBDV VP2 protein;

[0034] Figure 8 The hematocrit statistics of chickens immunized with recombinant virus H21001 after being challenged with strong CIAV virus;

[0035] Fig. 9 The statistical results of the bursal index of chickens immunized with the recombinant virus H21001 and challenged with the new variant of IBDV. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1: Construction and identification of recombinant HVT expressing CIAV VP1 and VP2 genes and IBDV VP2 gene

[0038] 1.1 Establishment of the reverse genetics operating system of HVTFC126 strain

[0039] The genomic DNA of HVT FC126 strain was extracted, and the genomic DNA of HVT FC126 strain was cloned into the pCC1Fos vector in segments according to the instructions of the CopyControl Fosmid Library ProductionKit kit. The GenBank accession number of the genomic DNA sequence of HVT FC126 strain is AF291866. The constructed recombinant cosmid was terminally sequenced using primers pCC1F and pCC1R (see Table 1 for details). According to the sequencing results, 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that cloned the genomic DNA fragments of HVT FC126 strain and could be spliced ​​to cover the complete HVT FC126 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.

[0040] 1.2 Construction and identification of recombinant cosmids expressing CIAV VP1 and VP2 genes

[0041] According to the coding sequences of VP1 and VP2 genes of CIAV JS15166 strain (GenBank accession number KY486153), the target gene CAVP12 (shown in SEQ ID NO.1) was synthesized by chicken codon optimization design, wherein the coding sequences of VP1 and VP2 genes were connected with the coding sequence of the self-cleaving peptide of porcine Teschovirus 2A (P2A). The target gene CAVP12 was obtained by PCR amplification using primers CAVP1EF and CAVP2CR, and the purified PCR product was cloned into the downstream of the chicken β-actin promoter of the pCAGGS vector through the EcoRI and ClaI restriction sites; at the same time, the post-transcriptional regulatory sequence of the woodchuck hepatitis virus was inserted between the target gene CAVP12 and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-CAVP12 that co-expresses the CIAV VP1 and VP2 genes. The recombinant plasmid was sequenced using primers CAGF and CAGR, and it was found that the cloned target gene sequence was correct.

[0042] According to the instructions of the Counter Selection BAC Modification Kit, the expression framework CAGW-CAVP12 (shown in SEQ ID NO.2) containing chicken β-actin promoter, CAVP12 gene sequence, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit β-globulin polyadenylation sequence was inserted into the recombinant cosmid HVT04 between nucleotides 95322-95323 of the HVT FC126 genome to construct the recombinant cosmid HVT04-53-CAVP12 that co-expresses CIAV VP1 and VP2 genes. The recombinant cosmid was identified by PCR using the target gene primer CAVP1EF and the downstream homology arm primer HVT54R. The results showed that the PCR fragment of about 3486 bp was obtained ( Figure 1 ), which was consistent with expectations. The sequencing results showed that the PCR product contained the CAVP12 gene sequence and the downstream homology arm sequence of the target gene, and the sequence was correct. The parental cosmid HVT04 had no target gene insertion sequence, and the PCR result was negative. The above results indicate that the recombinant cosmid HVT04-53-CAVP12 was constructed correctly.

[0043] 1.3 Construction and identification of recombinant cosmids expressing IBDV VP2 gene

[0044] According to the VP2 gene sequence of varIBDV FJ-18 strain (GenBank accession number OK167034), the target gene IBDVA2 (shown in SEQ ID NO.3) was synthesized after chicken codon optimization design, and the synthesized target gene IBDVA2 was cloned into plasmid pUC57, and the resulting recombinant plasmid was named pUC57-IBDVA2. The recombinant plasmid pUC57-IBDVA2 cloned with the IBDVA2 gene was used as a template, and the IBDVA2 gene was obtained by PCR amplification using primers IBDVA2F and IBDVA2R. The purified PCR product was cloned into the downstream of the mouse cytomegalovirus promoter of the pCMV vector to construct a recombinant plasmid pCMV-IBDVA2 expressing the varIBDV VP2 gene.

[0045] According to the instructions of the Counter Selection BAC Modification Kit, the expression framework CMV-IBDVA2 (shown in SEQ ID NO.4) containing the mouse cytomegalovirus promoter, the IBDV VP2 gene coding sequence and the SV40 polyadenylation sequence was inserted into the recombinant cosmid HVT05 between nucleotides 112071-112088 of the HVT FC126 genome to construct the recombinant cosmid HVT05-65-IBDVA2 expressing the IBDV VP2 gene. The recombinant cosmid was identified by PCR using the target gene primer IBDVA2F and the target gene downstream homology arm primer HVT65F. The results showed that a PCR fragment of about 1800 bp was obtained ( Figure 1 ), which was consistent with expectations. The sequencing results showed that the PCR product contained the varIBDVVP2 gene sequence and the downstream homology arm sequence of the target gene, and the sequence was correct. The above results showed that the recombinant cosmid HVT05-65-IBDVA2 was constructed correctly.

[0046] Table 1 PCR primers used to construct and identify recombinant cosmids

[0047]

[0048] 1.4 Rescue and identification of recombinant HVT co-expressing CIAV VP1, VP2 genes and IBDV VP2 gene

[0049] The recombinant cosmids HVT04-53-CAVP12, HVT05-65-IBDVA2 and the other four parental cosmids HVT01, HVT02, HVT03 and HVT06 cloned with the genome fragment of HVT FC126 strain were extracted using a plasmid extraction kit. The above six recombinant cosmids were co-transfected with the parental cosmids into CEF cells using the calcium phosphate transfection method. The viruses were harvested after 4-5 days of culture until plaque lesions appeared. The viruses were continuously passaged and preserved in CEF cells. The recombinant virus with the CAVP12 expression framework CAGW-CAVP12 inserted between nucleotides 95322-95323 of the HVT FC126 genome and the IBDV VP2 gene expression framework CMV-IBDVA2 inserted between nucleotides 112071-112088 was rescued and named H21001. The plaque lesions produced by the recombinant virus on CEF were as follows: Figure 2 shown.

[0050] The recombinant virus genomic DNA was extracted, and the parent virus HVT FC126 strain genomic DNA was used as a control. The recombinant virus was identified by PCR using the target gene specific primer CIAVP1EF and the target gene downstream homology arm primer HVT54R. The PCR results showed that the PCR product obtained by amplification of the recombinant virus was about 3486 bp in length, which was consistent with the expected size. Figure 3 As shown. The sequencing results showed that the PCR product contained the CAVP12 gene sequence and the downstream homology arm sequence of the target gene, and the sequence was correct. However, the parent virus HVT FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene expression framework CAGW-CAVP12 was correctly inserted into the genome of the HVT FC126 strain.

[0051] At the same time, the extracted recombinant viral genomic DNA was identified by PCR using the target gene primer IBDVA2F and the target gene downstream homology arm primer HVT65F, and the PCR product was further sequenced and analyzed. The PCR results showed that the PCR product obtained by recombinant virus amplification was about 1800bp in length, which was consistent with the expected size ( Figure 3 ). The sequencing results showed that the PCR product contained the varIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene, and the sequence was correct. The parent virus HVT FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene expression cassette CMV-IBDVA2 was correctly inserted into the genome of the HVTFC126 strain.

[0052] Example 2: In vitro biological characteristics analysis of recombinant HVT co-expressing CIAV VP1, VP2 genes and IBDV VP2 gene

[0053] 2.1 Detection of recombinant virus H21001 expressing CIAV VP1, VP2 proteins and IBDV VP2 protein

[0054] The recombinant virus H21001 and the parental virus HVT FC126 were inoculated into CEF cells cultured in 6-well plates, with 3 wells inoculated with each virus, and 3 uninoculated wells were set up as controls. After 3-4 days of culture, plaque lesions appeared and the cells were fixed with anhydrous ethanol. CIAV VP1, VP2 polyclonal antibodies and IBDV VP2 monoclonal antibodies were used as primary antibodies, FITC-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies, and indirect immunofluorescence assay was used to detect the expression of CIAV VP1, VP2 protein and IBDV VP2 protein, and CEF inoculated with the parental virus FC126 was used as a negative control. The results showed that cells infected with the recombinant virus H21001 could react with CIAV VP1, VP2 polyclonal antibodies and IBDV VP2 monoclonal antibodies, showing green fluorescence signals ( Figure 4 ). No fluorescence was observed in cells infected with the parental virus FC126 strain and in control cells that were not infected. The above results indicate that the recombinant virus H21001 can co-express CIAV VP1, VP2 proteins and IBDV VP2 proteins in infected cells.

[0055] 2.2 Analysis of in vitro replication characteristics of recombinant virus H21001

[0056] The recombinant virus H21001 and the parent virus HVT FC126 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 virus in vitro replication kinetic curve was drawn to analyze the in vitro replication characteristics of the recombinant virus H21001 and the parent virus HVT FC126 in CEF. The results showed that the replication titers of the recombinant virus H21001 and the parent virus HVT FC126 reached the highest peak at 120 hours after infection, which were 2.17×10 5 PFU / ml and 2.34×10 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 HVTFC126 (P>0.05)( Figure 5 ). The above results show that the recombinant virus H21001 has good replication ability in CEF and its in vitro replication characteristics are consistent with those of the parent virus HVT FC126 strain.

[0057] 2.3 Genetic stability test of recombinant virus H21001

[0058] The 5th generation recombinant virus H21001 was continuously passaged on CEF to the 20th generation. The 20th generation cytotoxic cells were selected, the viral genomic DNA was extracted, and PCR identification and sequencing were performed to detect the genetic stability of the target gene sequence in the recombinant virus genome. The results showed that the 20th generation recombinant virus was identified by PCR using the target gene specific primer CAVP1EF and the target gene downstream homologous arm primer HVT54R, and a fragment of about 3486 bp could be amplified, which was consistent with expectations ( Figure 6 ). The 20th generation recombinant virus genomic DNA was amplified and sequenced using primers CAGF and CAGR, and the inserted target gene CAVP12 sequence was found to be correct. The 20th generation recombinant virus was identified by PCR using the target gene primer IBDVA2F and the target gene downstream homology arm primer HVT65F, and a fragment of about 1800 bp was amplified, which was consistent with expectations ( Figure 6 ). The sequencing results showed that the PCR product contained the varIBDV VP2 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 target genes CIAV VP1, VP2 genes and IBDV VP2 gene inserted into the HVT genome can stably exist during the passage process.

[0059] At the same time, the 20th generation recombinant virus was inoculated into CEF cells, and indirect immunofluorescence test was performed with CIAV VP1, VP2 polyclonal antibodies and IBDV VP2 monoclonal antibodies to detect the stability of target gene expression. The results showed that after the recombinant virus H21001 was continuously propagated on CEF cells to the 20th generation, the CIAV VP1, VP2 genes and IBDV VP2 genes inserted into the HVT genome could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus H21001 has good genetic stability.

[0060] Example 3: Safety and immunogenicity testing of recombinant virus H21001

[0061] 3.1 Safety testing of recombinant virus H21001

[0062] The fifth-generation recombinant virus H21001 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 H21001 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 virus H21001 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 H21001 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 H21001 is safe for SPF chickens.

[0063] 3.2 Immunoprotective test of recombinant virus H21001 against CIAV and IBDV

[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 H21001 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parent virus HVT FC126 at the same dose, and Group 3 was not immunized as a blank control group. 21 days after immunization, 10 chickens were respectively challenged with the virulent CIAV SD23 strain; 14 days after challenge, the clinical symptoms and survival of the test chickens were recorded; 14 days after challenge, blood was collected to detect the hematocrit value of the test chickens, and the hematocrit value was less than 27% and was judged to be onset. 28 days after immunization, the remaining 10 chickens in Group 1 and Group 2 were challenged with the new variant strain FJ of IBDV, and 7 days after challenge, the clinical symptoms of the test chickens were recorded, and the survival was counted; 7 days after challenge, the bursal lesions were observed and the bursal weight ratio (F / B) and bursal 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; BBIX less than 0.7 was judged as bursa atrophy. Group 3 was not challenged with poison and served as the healthy control group.

[0065] The results showed that after the parental virus HVT FC126 vaccination group was challenged with strong CIAV virus, 9 of the 10 chickens became ill, and the hematocrit value was less than 27% below the normal level. After the recombinant virus H21001 vaccination group was challenged with strong CIAV virus, all 10 chickens survived healthily, without obvious adverse clinical symptoms, and the hematocrit values ​​were all greater than 27%. After the parental virus FC126 vaccination group was challenged with the new variant of IBDV, the bursa of Fabricius of all 10 chickens showed atrophic lesions, and BBIX was less than 0.7. After the recombinant virus H21001 immunization group was challenged with the new variant of IBDV, all 10 chickens survived healthily, among which 9 chickens had no obvious lesions in the bursa of Fabricius, and BBIX was greater than 0.7; the bursa of 1 chicken was slightly atrophied, and BBIX was less than 0.7 ( Fig. 9 The above results show that the recombinant virus H21001 has a good immune protection effect against both the virulent CIAV and the virulent new variant strains of IBDV.

Claims

1. A recombinant turkey herpesvirus (HVT) co-expressing the VP1 and VP2 genes of chicken infectious anemia virus (CIAV) and the VP2 gene of infectious bursal disease virus (IBDV), characterized in that: The recombinant turkey herpes virus is obtained by inserting the expression frame CAGW-CAVP12 containing chicken β-actin promoter, CIAVVP1 and VP2 gene coding sequences, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence into the 95322-95323 nucleotides of the HVT genome, and inserting the expression frame CMV-IBDVA2 containing mouse cytomegalovirus promoter, IBDVVP2 gene coding sequence and SV40 polyadenylation sequence into the 112071-112088 nucleotides of the HVT genome.

2. The recombinant turkey herpes virus strain according to claim 1, characterized in that The CIAVVP1 and VP2 gene coding sequences are obtained by connecting the VP1 and VP2 gene coding regions with the porcine Teschovirus 2A self-cleaving peptide coding sequence, and the nucleotide sequence thereof is shown in SEQ ID NO.

1.

3. The recombinant turkey herpes virus strain according to claim 2, characterized in that The expression framework CAGW-CAVP12 sequentially comprises chicken β-actin promoter, CIAVVP1 and VP2 gene coding sequences, woodchuck hepatitis virus post-transcriptional regulatory sequences and rabbit β-globulin polyadenylic acid sequences. The nucleotide sequence of the expression framework CAGW-CAVP12 is shown in SEQID NO.

2.

4. The recombinant turkey herpes virus strain according to claim 1, characterized in that The IBDVVP2 gene coding sequence is synthesized based on the VP2 gene sequence of the varIBDVFJ-18 strain with GenBank accession number OK167034 after chicken codon optimization design, and its nucleotide sequence is shown in SEQ ID NO.

3.

5. The recombinant turkey herpes virus strain according to claim 1, characterized in that The IBDV is a novel variant of chicken infectious bursal disease virus (varIBDV).

6. The recombinant turkey herpes virus strain according to claim 1, characterized in that The nucleotide sequence of the expression framework CMV-IBDVA2 is shown in SEQ ID NO.

4.

7. The recombinant turkey herpes virus strain according to claim 1, characterized in that The HVT is HVT FC126 strain, and the GenBank accession number of the genome sequence 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 following steps are involved: (1) Establishment of the multi-fragment cosmid rescue system of HVT 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 HVT genome, were selected; among them, HVT01 contained the nucleotide fragment of 1-30189 of the HVT FC126 genome, HVT02 contained the nucleotide fragment of 21572-61476 of the HVT FC126 genome, HVT03 contained the nucleotide fragment of 50871-90478 of the HVT FC126 genome, HVT04 contained the nucleotide fragment of 73762-108139 of the HVT FC126 genome, HVT05 contained the nucleotide fragment of 96776-135815 of the HVT FC126 genome, and HVT06 contained the nucleotide fragment of 14777-161117 of the HVT FC126 genome. The nucleotide fragment from position 129602 to 159160 of the FC126 genome; (2) Construction of recombinant cosmids expressing CIAVVP1 and VP2 genes A recombinant expression plasmid comprising a chicken β-actin promoter, CIAVVP1 and VP2 gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence expression framework CAGW-CAVP12 was constructed, wherein the VP1 and VP2 gene coding regions were connected by a porcine Teschovirus 2A self-cleaving peptide coding sequence; the expression framework CAGW-CAVP12 was cloned into the 95322-95323 nucleotides of the HVT FC126 genome in the recombinant cosmid HVT04 by using the Red / ET recombination method, and a recombinant cosmid that co-expressed CIAVVP1 and VP2 genes was constructed and named HVT04-53-CAVP12; (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-IBDVA2 was constructed; the expression frame CMV-IBDVA2 was cloned into the recombinant cosmid HVT05 between nucleotides 112071 and 112088 of the HVT FC126 genome using the Red / ET recombination method to construct a recombinant cosmid expressing the IBDVVP2 gene, named HVT05-65-IBDVA2; (4) Rescue of recombinant HVT co-expressing CIAV VP1 and VP2 genes and varIBDV VP2 gene The recombinant cosmids HVT04-53-CAVP12, HVT05-65-IBDVA2 and the other four parental cosmids HVT01, HVT02, HVT03 and HVT06 cloned with the genomic fragments of HVT FC126 strain were extracted; 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. The recombinant virus in which the expression framework CAGW-CAVP12 was inserted between nucleotides 95322-95323 of the HVT FC126 strain genome and the expression framework CMV-IBDVA2 was inserted between nucleotides 112071-112088 of the HVT FC126 strain genome was rescued and named H21001.

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

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