Recombinant Marek's disease virus for expressing IBDV (Infectious Bursal Disease Virus) VP2 and FAdV-4 Fiber2 genes as well as construction method and application of recombinant Marek's disease virus

Abstract

The invention discloses a recombinant Marek's disease virus for expressing IBDV (Infectious Bursal Disease Virus) VP2 and FAdV-4 Fiber2 genes as well as a construction method and application of the recombinant Marek's disease virus, and belongs to the technical field of biology. The recombinant Marek's disease virus is prepared by the following steps: inserting a gene segment CMV-VP2 containing a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence into a US2 gene of a Marek's disease virus Meq gene deleted vaccine strain; a gene segment SV40-Fiber2 containing an SV40 promoter sequence, an FAdV-4 Fiber2 gene and a terminator sequence is inserted into a UL41 gene of a Marek's disease virus Meq gene deleted vaccine strain, and a recombinant virus capable of simultaneously expressing an IBDV VP2 gene and the FAdV-4 Fiber2 is obtained. After chickens are immunized by the recombinant virus, vvIBDV and FAdV-4 can be effectively protected. The invention provides a technical means for developing the recombinant MDV live vector vaccine for simultaneously expressing the IBDV VP2 protein and the FAdV-4 Fiber2 protein.

Description

Technical Field

[0001] The present invention relates to a recombinant Marek's disease virus and a construction method and application thereof, and in particular to a recombinant Marek's disease virus expressing the VP2 gene of chicken infectious bursal disease virus and the Fiber2 gene of serum type 4 avian adenovirus and a construction method and application thereof. The present invention belongs to the field of biotechnology. Background Art

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive, and fatal infectious disease caused by the infectious bursal disease virus (IBDV). It mainly affects chicks and is listed as a "disease of major socioeconomic importance" by the Office International des Epizooties (OIE). Since its first outbreak in the United States in 1957, the disease has become widespread in Europe, Southeast Asia, Africa, and South America, and has caused a serious epidemic in China, posing a continuous threat to the poultry industry. In recent years, the emergence of very virulent strains of IBDV (vvIBDV) and antigenic variants has further increased the hazard of the disease. The mortality rate of vvIBDV exceeds 60%, and resistant chickens may fail to be immunized against vaccines such as avian influenza and Newcastle disease.

[0003] IBDV belongs to the BiRNA virus family. Its genome consists of two double-stranded RNA segments. The VP2 protein encoded by the A segment is the main capsid protein and host protective antigen of IBDV. Vaccine immunization is the main means of preventing and controlling vvIBDV, but the strong pathogenicity and inadaptability to cell culture of vvIBDV mean that the vaccine strain can only be obtained by attenuating it through subculture in chicken embryos or CEF cells. This traditional method is time-consuming and the results are random. In the face of highly pathogenic and easily mutated vvIBDV, especially in the event of an outbreak, there is an urgent need to quickly construct a new vaccine. At the same time, the widespread use of existing IBD attenuated vaccines and moderately toxic vaccines has led to immune suppression in chicken flocks and increased viral mutation. Therefore, it is particularly urgent to develop a safer and more effective new IBD vaccine.

[0004] Hydropericardium hepatitis syndrome (HHS) is an acute, highly contagious and immunosuppressive infectious disease caused by avian adenovirus serotype 4 (FAdV-4). In 1987, FAdV-4 was first discovered in the Angara region of Pakistan and then spread to the world. Since 2015, FAdV-4 has frequently broken out in China, with a mortality rate of up to 100% in chicks, causing serious economic losses. After chickens are infected with FAdV-4, they can be seen clinically with ruffled feathers and depression, and autopsy shows yellowish effusion in the pericardial sac accompanied by hepatitis and nephritis. The FAdV-4 genome is about 45 kb, mainly encoding Fiber1, Fiber2, Penton base, and Hexon structural proteins. Among them, Fiber2 protein plays a key role in the function of virus recognition and invasion of host cells. Fiber2 has good immunogenicity, and studies have shown that using Fiber2 as a subunit vaccine to immunize animals can provide 100% protection against FAdV-4 attack. The main challenges of existing HHS inactivated vaccines and subunit vaccines are the short duration of immune protection and the need for repeated vaccination. Therefore, the development of a new genetically engineered vaccine with long-lasting immune effects and broad-spectrum protection is crucial to effectively control HHS.

[0005] Marek's disease (MD) is an infectious tumor disease caused by Marek's disease virus (MDV). Due to its high infectiousness and tumorigenicity, MD has become one of the major infectious diseases facing the global poultry industry. MDV is divided into three serotypes, and the main pathogenic wild strain is currently serotype 1 (MDV 1). The Meq gene-deficient vaccine strain rMSΔmeq is based on the parental strain MDV MS strain, but lacks the first half of the 468bp base sequence of the main tumorigenic gene Meq gene. Its genome is large and provides multiple replication non-essential regions for the insertion of exogenous genes, making it an ideal vector for constructing recombinant live vaccines. The present invention is based on rMSΔmeq, utilizes the multi-fragment cosmid rescue system and Red / ET recombination technology, inserts the IBDV VP2 gene expression cassette and the FAdV-4 Fiber2 gene expression cassette into the rMSΔmeq genome, successfully constructs the recombinant virus rMDV-VP2-Fiber2 expressing the IBDV VP2 and FAdV-4 Fiber2 genes, and evaluates its biological activity and immune efficacy. The evaluation results show that SPF chickens inoculated with rMDV-VP2-Fiber2 can provide good protection against vvIBDV and FAdV-4. Summary of the invention

[0006] The object of the present invention is to provide a recombinant Marek's disease virus expressing the VP2 gene of infectious bursal disease virus of chickens and the Fiber2 gene of serotype 4 avian adenovirus, and a construction method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical means: The invention discloses a recombinant Marek's disease virus expressing IBDV VP2 and FAdV-4 Fiber2 genes, wherein a gene fragment CMV-VP2 comprising a CMV promoter sequence, an infectious bursal disease virus VP2 gene and a terminator sequence is inserted into the Marek's disease virus Meq Gene-deficient vaccine strain US2 The gene fragment SV40-Fiber2 containing the SV40 promoter sequence, the serotype 4 fowl adenovirus fiber 2 gene and the terminator sequence was inserted into the Marek's disease virus Meq Gene-deficient vaccine strains UL41 Within the gene, a recombinant Marek's disease virus strain was obtained that simultaneously expressed the IBDV VP2 gene and FAdV-4 Fiber2.

[0008] Wherein, preferably, the Marek's disease virus Meq The gene-deficient vaccine strain is the rMSΔMeq strain, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and its microbial deposit number is: CGMCC No.4612.

[0009] Preferably, the recombinant Marek's disease virus is a gene fragment CMV-VP2 comprising a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence inserted into a Marek's disease virus. Meq Gene-deleted vaccine strain rMSΔMeq US2 The nucleotide sequence at positions 156501-157116 of the rMSΔMeq strain genome sequence was replaced. At the same time, the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, the FAdV-4 Fiber2 gene and the terminator sequence was inserted into the rMSΔMeq strain. UL41 The nucleotide sequence at positions 96927-97106 of the rMSΔMeq strain genome sequence was replaced to obtain a recombinant Marek's disease virus strain that simultaneously expressed the IBDV VP2 gene and the FAdV-4 Fiber2 gene.

[0010] Among them, preferably, the VP2 gene is from the infectious bursal disease virus HLJ0504 strain, and the Fiber2 gene is from the serotype 4 avian adenovirus virulent strain HLJFAd15 strain, whose GenBank sequence number is KU991797.

[0011] Among them, preferably, the nucleotide sequence of the gene fragment CMV-VP2 is shown as SEQ ID NO.15, and the nucleotide sequence of the gene fragment SV40-Fiber2 is shown as SEQ ID NO.14.

[0012] Furthermore, the present invention also proposes a method for constructing the recombinant Marek's disease virus, comprising the following steps: (1) Marek's disease virus Meq Construction of a reverse genetics operating system for the gene-deleted vaccine strain rMSΔMeq The reverse genetics operating system comprises 5 cosmids, which are Marek's disease virus Meq The gene fragment of the gene-deficient vaccine strain rMSΔMeq was cloned into pCC1Fos. The five cosmids contain Marek's disease virus Meq The nucleotide sequences of the genome of the gene-deficient vaccine strain rMSΔMeq strain at positions 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526 are named pMS-1, pMS-2, pMS-3, pMS-4 and pMS-5, respectively, wherein pMS-3 comprises the UL41 gene of the rMSΔMeq strain, and pMS-4 comprises the US2 gene of the rMSΔMeq strain; the Marek's disease virus Meq The genome sequence GenBank accession number of the gene-deleted vaccine strain rMSΔMeq is JQ314003.1; (2) Construction and identification of recombinant cosmids pMS-VP2 and pMS-Fiber2 expressing target genes The pMS-3 cosmid and the pMS-4 cosmid were respectively transfected into competent cells containing the pRed E / T expression plasmid to prepare the corresponding competent cells; the rpsl-neo expression cassette DNA was used as a template, and the primers UL41-rpsl F / R and US2-rpsl F / R were used to amplify the rpsl-neo gene fragment containing the homologous arms at both ends of the UL41 insertion site and the homologous arms at both ends of the US2 insertion site, respectively, which were purified and transfected into competent cells containing pMS-3 and pMS-4 to prepare the competent cells; the IBDV VP2 gene expression plasmid was used as a template, and the primers US2-CMV F / R were used to amplify the CMV-VP2 gene fragment containing the homologous arms at both ends of the US2 insertion site, respectively, which were purified and electroporated into the rpsl-neo competent cells containing the homologous arms at both ends of the US2 insertion site; the FAdV-4 Fiber2 gene expression plasmid was used as a template, and the primers UL41-SV40 F / R was amplified by PCR to obtain the Fiber2 gene fragment containing the homologous arms at both ends of the UL41 insertion site, which was purified and electroporated into the competent cells of rpsl-neo containing the homologous arms at both ends of the UL41 insertion site; positive colonies were picked and expanded for culture and then the cosmids were extracted, and the obtained cosmids were named pMS-VP2 and pMS-Fiber2 respectively; the names and sequences of the primers required for construction are as follows: ; (3) Rescue of recombinant Marek's disease virus rMDV-VP2-Fiber2 The recombinant cosmids pMS-VP2 and pMS-Fiber2 and the other three parental cosmids pMS1, pMS2 and pMS5 cloned with rMSΔmeq genomic fragments were extracted using a plasmid extraction kit. The five cosmids were purified by the calcium phosphate method. Note The linearized cosmids were co-transfected into CEF cells. After 4-5 days, the transfected cells were observed for the appearance of MDV-specific plaques. After the appearance of plaques, the viruses were harvested and frozen in liquid nitrogen. The genomic DNA of the recombinant virus was extracted and identified by PCR and sequencing. The correctly identified recombinant MDV was named rMDV-VP2-Fiber2.

[0013] Among them, preferably, the IBDV VP2 gene expression plasmid is obtained by amplifying the VP2 gene using the genome of the IBDV HLJ-0504 strain as a template and then cloning it into the CMV expression box of the vector pEGFP-N1; the FAdV-4 Fiber2 gene expression plasmid is obtained by amplifying the Fiber2 gene using the genome DNA of the FAdV-4 virulent strain HLJFAd15 as a template and then cloning it into the BstB Ⅰ and Sma Ⅰ sites of the pCDNA3.1 vector to obtain the plasmid pSV40-Fiber2; The nucleotide sequence of the gene fragment CMV-VP2 containing the CMV promoter sequence, IBDV VP2 gene and terminator sequence in the IBDV VP2 gene expression plasmid is shown in SEQ ID NO.15; the nucleotide sequence of the gene fragment SV40-Fiber2 containing the SV40 promoter, FAdV-4 Fiber2 gene and terminator sequence in the FAdV-4 Fiber2 gene expression plasmid is shown in SEQ ID NO.14.

[0014] Furthermore, the present invention also proposes the use of the recombinant Marek's disease virus in the preparation of a drug for preventing FAdV-4 and / or IBDV infection.

[0015] Among them, preferably, the IBDV is a very virulent strain of IBDV.

[0016] Among them, preferably, the drug is a recombinant Marek's disease virus live vector vaccine expressing IBDV VP2 gene and FAdV-4 Fiber2 gene.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses recombinant cloning technology to insert the gene fragment CMV-VP2 containing the VP2 gene of chicken infectious bursal disease virus and the CMV promoter sequence into the US2 gene of the Marek's disease virus gene-deficient vaccine strain (rMS-ΔMeq), and successfully constructs a recombinant cosmid containing the CMV-VP2 expression frame. At the same time, we also insert the gene fragment SV40-Fiber2 containing the Fiber2 gene of serotype 4 avian adenovirus and the SV40 promoter sequence into the UL41 gene of the Marek's disease virus gene-deficient vaccine strain (rMS-ΔMeq), and construct a recombinant cosmid containing the SV40-Fiber2 expression frame. These two recombinant cosmids and the other three cosmids containing the rMS-ΔMeq genome are co-transfected into CEF cells to rescue and obtain the recombinant Marek's disease virus rMDV-VP2-Fiber2 expressing the VP2 gene of chicken infectious bursal disease virus and the Fiber2 gene of serotype 4 avian adenovirus. The results of the study showed that rMDV-VP2-Fiber2 not only maintained the same in vitro replication ability as the parent strain rMS-Δmeq, but also exhibited good genetic stability. In addition, after rMDV-VP2-Fiber2 immunized chickens, it was able to provide effective protection against vvIBDV and FAdV-4. The invention provides a technical means for developing a recombinant MDV live vector vaccine that simultaneously expresses IBDV VP2 protein and FAdV-4 Fiber2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] tú1This is the result of PCR identification of recombinant cosmids pMS-Fiber2 and pMS-VP2; tú2 The cytopathic effect of recombinant Marek's disease virus rMDV-VP2-Fiber2 on CEF is shown; tú3 This is a diagram showing the PCR identification results of the recombinant Marek's disease virus rMDV-VP2-Fiber2 genomic DNA; tú4 This is the result of indirect immunofluorescence assay to detect the expression of VP2 protein and Fiber2 protein in CEF infected with recombinant Marek's disease virus rMDV-VP2-Fiber2 and parental virus rMSΔMeq; tú5 It is the replication kinetic curve of the recombinant Marek's disease virus rMDV-VP2-Fiber2 and the parental virus rMSΔMeq on CEF; tú6 This is a graph showing the immune protection results of chickens immunized with the recombinant Marek's disease virus rMDV-VP2-Fiber2 and the parental virus rMSΔMeq against the challenge of FAdV-4 strain; tú7 This is a graph showing the immune protection results of chickens immunized with recombinant Marek's disease virus rMDV-VP2-Fiber2 and parental virus rMSΔMeq against IBDVHLJ-0504 strain attack. DETAILED DESCRIPTION

[0019] The present invention is further described below by way of examples, which are intended only to provide a better understanding of the present invention and are not intended to limit the scope of the present invention. Except for special examples, the following examples are all conventional experimental methods and operating procedures in the art.

[0020] Example 1 Marek's Disease Virus Meq Construction of gene-deleted vaccine strain rMSΔMeq Marek's disease virus Meq The gene-deficient vaccine strain rMSΔMeq is described in the publication number CN102363769B, and the invention name is "Chicken Marek's Disease Virus Meq The patent application for "Gene-deficient vaccine strain, construction method and application thereof" is in the GenBank accession number JQ314003.1. The specific construction method is as follows: 1. Construction of recombinant virus transfer plasmid: 1. Test methods 1) Primer design and synthesis: Referring to the published gene sequence of MDV virulent GA strain (No.AF147806), primer pairs Primer A and Primer B were designed for MeqFragments A and B on both sides of the gene are used as homologous sequences for constructing transfer vector plasmids. The upstream of primer pair LacZB is on the LacZ gene, and the downstream is on the recombination arm B, which is used to identify whether there is recombination; the upstream of primer pair ALB is on the recombination arm A, and the downstream is on the recombination arm B, which is used to identify the location and number of deletions. If the amplified size of primer ALB is 660 bp, it indicates Meq The gene is not deleted. If the amplified size is 4288 bp, it indicates that LacZ is inserted into the expected position. Meq The upstream and downstream are located at Meq The two sides of the gene are used for PCR identification of the recombinant vector rMS-LacZΔMeq. For the detection of the parental virus MS strain, Meq The gene is intact, so the amplified fragment size is about 1.4Kb. For the recombinant strain rMSΔMeq, 468bp (shown in SEQ ID NO.1) is missing, and the amplified result is about 1kb. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The primer names and sequences are shown in Table 1: ; 2) Preparation of DNA template: Chicken embryo fibroblasts (CEF) were prepared from 9-day-old SPF chicken embryos according to conventional methods. The MDV MS strain was inoculated into CEF and cultured in a 37°C 5 mL / L CO2 incubator. When the pathological rate reached 80%, the cells were digested and harvested, and total DNA was extracted according to conventional methods and dissolved with an appropriate volume of TE (pH7.4).

[0021] 3) Amplification and recovery of homologous fragments: The total viral genome DNA extracted above was used as a template for amplification. The reaction system was 25 μL: 17.5 μL of ultrapure water, 2.5 μL of 10×PCR reaction buffer, 1 μL of dNTP (10 mmol∙L-1), 1 μL of upstream and downstream primers (10 pmol∙L-1), 1 μL of rTaq enzyme (5 U∙μL-1), and 2 μL of DNA (0.5 μg∙μL-1). The two PCR reaction conditions were: denaturation at 94°C for 5 min, then denaturation at 94°C for 1 min, annealing at 57°C (annealing of B fragment at 58°C) for 1 min, extension at 72°C for 1 min, 30 cycles, and finally extension at 72°C for 10 min. The amplified products were identified by 1.0% agarose electrophoresis containing ethidium bromide (EB). The correctly identified products were recovered by fragment gel.

[0022] 4) Construction of transfer vector plasmid: Using genomic DNA of MDV MS strain as template, the transfer vector plasmid was obtained by PCR method. MeqThe homology arm A of the upstream fragment of the gene was digested with SalⅠ and SphⅠ and connected to pUC19 to obtain plasmid pUCA; the B fragment obtained by PCR amplification was digested with SalⅠ and SacⅠ and connected to pUCA to obtain plasmid pUCAB. The LacZ expression gene cassette obtained by SalⅠ digestion was connected to the pUCAB plasmid to obtain plasmid pALacZB. The plasmid was extracted with Qiagen plasmid extraction kit, dissolved in TE with pH 7.4, and stored at -20℃ for later use.

[0023] II. Acquisition and identification of recombinant virus - rMS△Meq First recombination: 1) Co-transfection: MDV MS strain genomic total DNA and transfer vector plasmid pALacZB were co-transfected into CEF using calcium phosphate precipitation method. The specific method is as follows: chicken embryo fibroblasts were prepared. When CEFs grew into a monolayer within 24 hours, they were digested and subcultured into cell culture dishes with a diameter of 60 mm. The CEF inoculation amount was 9×10 6 First, add 388μL of nuclease-free deionized water to a clean and sterile 1.5mL centrifuge tube; then add 10~18µL of the prepared MS strain infected CEF genome total DNA (0.8 μg∙μL-1) and 2 μL of plasmid pALacZB (1 μg∙μL-1); add 30~36 μL TE (pH 7.4); slowly add 62μL 2mo1∙L-1 CaCl2 and mix slowly; use a 1mL pipette to slowly add 500μL of 2×HEPES from the bottom, use a 200μL micropipette to blow about 20 bubbles from the bottom to mix it, and place it in a 37℃ incubator for 30 minutes, during which it will be packaged into small precipitated particles; gently blow and resuspend the calcium phosphate DNA, evenly add it to the surface of the prepared CEF secondary cells, and gently shake the cell culture dish several times. At this time, the culture medium turns yellow-orange and is placed at 37℃5% CO2 incubator for 2-3 hours; discard the upper culture medium, rinse with serum-free culture medium or sterile PBS buffer 3 times, add 2 mL of 15% glycerol shock solution to shock for 90 seconds, and then rinse with serum-free culture medium or sterile PBS buffer 3 times; add 3% fetal bovine serum culture medium to promote cell growth into a monolayer, and culture in a 37℃ 5% CO2 incubator. The whole process is operated under sterile conditions. After culturing for about 4-6 days, when typical Marek's plaques appear, proceed to the next step of cloning and purification.

[0024] 2) Plaque cloning and purification of recombinant virus: After plaques appear, place them under an inverted microscope for observation in order to prevent missed detection. Pour out the culture medium and add 2mL of 3% maintenance solution, add 20μL of X-Gal (20mg∙mL-1) to make the total concentration 0.2mg∙mL-1, and culture at 37°C, 5% CO2 for 2h. Select blue plaques for digestion, cloning and purification. Add 0.25% trypsin to the circled plaques, digest for several minutes, scratch with the tip of a gun a few times, suck up the digestion solution, add to the chicken embryo fibroblasts that have grown into a monolayer, suck out some of the maintenance solution from the plate with monolayer cells, wash the circled and digested plaques, add to the chicken embryo fibroblasts that have grown into a monolayer, continue to culture at 37°C, 5% CO2, and purify again after the plaques grow. After cloning, all plaques are blue.

[0025] 3) Identification of recombinant virus: The recombinant virus purified from plaques was inoculated on primary CEF. When the plaques grew to 70%, the cells were harvested and total DNA was extracted according to the molecular cloning method. LacZB primers and ALB primers were used for amplification respectively. The reaction system was 25μL: ultrapure water to 17 μL, 0×PCR reaction buffer 2.5μL, dNTP (10mmol∙L-1) 1 μL, upstream and downstream primers (10pmol) 1μL each, rTaq enzyme (5U∙μL-1) 1 μL, recombinant virus DNA 2μL (0.5μg∙μL-1). The reaction conditions for both PCR were: denaturation at 94℃ for 5min, then denaturation at 94℃ for 1min, annealing at 57℃ (annealing temperature of primer ALB is 58℃) ​​for 1min, extension at 72℃ (extension time of primer ALB is 4min) for 1min, 30 cycles, and finally extension at 72℃ for 10min. The PCR product was connected to the pMD-18T vector, and the bacterial solution was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing and further identification. The purified virus was named: rMS-LacZΔMeq. The recombinant virus rMS-LacZΔMeq was detected by PCR using primer LacZB, and a band consistent with the expected size (1300bp) was obtained.

[0026] Meq The gene deletion copy number was identified using primer ALB. The amplified size of the parental MS strain was about 656 bp, and the recombinant rMS-LacZΔMeq was about 4388 bp. The results showed that the size was consistent with the expected size, indicating that the construction was correct. It also proved that the two Meq The genes are missing.

[0027] Second recombination: The method is the same as the first recombination. The total genomic DNA of MDV rMS-LacZΔMeq strain and the transfer vector plasmid pUCAB are used for co-transfection to obtain the deletion MeqAnd the recombinant virus rMSΔMeq strain without LacZ.

[0028] Identification method of rMSΔMeq virus strain: The total DNA of rMSΔMeq infected CEF was extracted according to the conventional method. Meq The fragment size of the parental virus MS strain F5 was 1.4Kb, and the recombinant virus rMSΔMeq strain F25 was 1Kb, with no 1.4Kb band. The results were consistent with expectations, proving that the two recombinant viruses Meq The genes have been deleted. The sequencing results show Meq The first 468 bp of the gene ORF were deleted, which was consistent with the expected results of the experimental design.

[0029] Marek's disease virus Meq The gene-deficient vaccine strain rMSΔMeq was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the microbial deposit number is: CGMCC No.4612.

[0030] Example 2 Construction and Identification of Recombinant Marek's Disease Virus Expressing IBDV VP2 and FAdV-4 Fiber2 Genes 1. Materials and methods: 1.1 Plasmids, strains and viruses Marek's disease virus rMS-Δmeq strain (prepared in Example 1), chicken infectious bursal disease virus HLJ0504 strain (recorded in the following literature: Gao Li, Qi Xiaole, Gao Yulong, Gao Honglei, Qin Liting, Deng Xiaoyun, Yuwen Yanqing, Wang Xiaomei. Complete genome cloning and new feature analysis of infectious bursal disease virus super virulent strain HLJ0504. Chinese Journal of Preventive Veterinary Medicine. 2010, 32(5): 401-404. And publication number CN102973952A, invention name " The DNA vaccine expressing the polyprotein gene VP243 of infectious bursal disease virus and its construction method and application" (Chinese patent application) and the virulent serotype 4 avian adenovirus strain HLJFAd15 (GenBank sequence number KU991797) were preserved and provided by the Avian Immunosuppression Disease Research Group of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the plasmid pRed / ET expressing the recombinase and the plasmid containing the selection gene rpsl-neo were from the Counter-Selection BAC Modification Kit; the EPI300-T1 strain and the DH10B strain were preserved by our laboratory.

[0031] 1.2 Main Reagents Cell culture medium and fetal bovine serum were purchased from Gibco; conventional PCR reaction enzyme, restriction endonuclease, and ligase were purchased from TaKaRa; calcium phosphate transfection reagent was purchased from Invitrogen; plasmid extraction kit was purchased from QIAGEN; body fluid viral DNA / RNA small-scale preparation kit was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd.; cell lysis buffer was purchased from Biotech; IRDye-800-labeled goat anti-mouse IgG was purchased from Licor; mouse anti-rabbit IgG-FITC, rabbit anti-chicken IgG-HRP, and β-actin monoclonal antibody were purchased from Sigma; gI monoclonal antibody, Fiber2 protein, Fiber2 polyclonal antibody, and VP2 monoclonal antibody were prepared and preserved by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0032] 1.3 Marek's disease virus Meq Construction of a reverse genetics operating system for the gene-deleted vaccine strain rMSΔMeq The reverse genetics operating system comprises the following: Meq Five cosmids containing nucleotide sequences at positions 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526 of the genome of the gene-deficient vaccine strain rMSΔMeq were prepared by using the Copy Control Fosmidlibrary production kit to clone the corresponding Marek's disease virus. Meq The gene fragments of the gene-deficient vaccine strain rMSΔMeq were cloned into pCC1Fos and named pMS-1, pMS-2, pMS-3, pMS-4 and pMS-5 respectively; pMS-3 contained the UL41 gene of the rMSΔMeq strain, and pMS-4 contained the US2 gene of the rMSΔMeq strain; the Marek's disease virus Meq The GenBank accession number of the genome sequence of the gene-deleted vaccine strain rMSΔMeq is JQ314003.1.

[0033] Marek's disease virus Meq The GenBank accession number of the genome sequence of the gene-deleted vaccine strain rMSΔMeq is JQ314003.1.

[0034] 1.4 Construction of pSV40-Fiber2 expression plasmid The genomic DNA of the virulent FAdV-4 strain HLJFAd15 was used as a template to amplify the Fiber2 gene fragment using PCR primers SV40-Fiber2 F / R, and then the Fiber2 fragment was ligated into the pCDNA3.1 vector. BstB Ⅰ and Sma I site, and obtained plasmid pSV40-Fiber2, which was sequenced and identified. The nucleotide sequence of the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, FAdV-4 Fiber2 gene and terminator sequence is shown in SEQ ID NO.14.

[0035] .

[0036] 1.5 Construction of pCMV-VP2 expression plasmid Using the genomic DNA of the IBDV super-virulent strain HLJ0504 as a template, PCR primers CMV-VP2 F / R were used to amplify the VP2 gene fragment, and then the VP2 fragment was connected to the Nhe Ⅰ and Not Ⅰ sites of the pEGFP-N1 vector to obtain the plasmid pCMV-VP2, which was sequenced and identified. The nucleotide sequence of the gene fragment CMV-VP2 containing the CMV promoter sequence, the IBDV VP2 gene and the terminator sequence is shown in SEQ ID NO.15.

[0037] .

[0038] 1.6 Construction of recombinant cosmids expressing VP2 and Fiber2 genes The recombinant cosmid was constructed using a two-step Red recombination method: the recombinase plasmid pRed / ET was electrotransformed into competent cells DH10B, and positive colonies were picked to prepare pRed E / T-expressing electrotransformed competent cells DH10B, and then pMS-3 and pMS-4 cosmids were electrotransformed into competent cells DH10B containing pRed E / T expression plasmids, and positive colonies were picked to prepare competent cells. The plasmid containing the screening gene rpsl-neo was used as a template, and primers UL41-rpsl F / R and US2-rpsl F / R were used to amplify the rpsl-neo gene fragments containing homologous arms at both ends of the UL41 insertion site and homologous arms at both ends of the US2 insertion site, respectively. After purification, they were electrotransformed into competent cells containing pMS-3 and pMS-4, and positive colonies were picked to prepare competent cells. Using the IBDV VP2 expression plasmid pMS-VP2 as a template, primers US2-CMV F / R were used to amplify the VP2 gene fragments containing the homologous arms at both ends of the US2 insertion site by PCR, which were purified and electrotransferred to the competent cells of rpsl-neo containing the homologous arms at both ends of the US2 insertion site. Using the FAdV-4 Fiber2 gene expression plasmid pSV40-Fiber2 as a template, primers UL41-SV40 F / R were used to amplify the Fiber2 gene fragments containing the homologous arms at both ends of the UL41 insertion site by PCR, which were purified and electrotransferred to the competent cells of rpsl-neo containing the homologous arms at both ends of the UL41 insertion site. The positive colonies were picked and expanded for culture, and the clay was extracted. The obtained clays were named pMS-VP2 and pMS-Fiber2, respectively. The names and sequences of the primers required for construction are shown in Table 2.

[0039] ; 1.7 Rescue and identification of recombinant MDV The recombinant cosmids pMS-VP2 and pMS-Fiber2 and the other three parental cosmids pMS1, pMS2 and pMS5 cloned with rMSΔmeq genomic fragments were extracted using a plasmid extraction kit. The five cosmids were purified by the calcium phosphate method. Note I. The linearized cosmids were co-transfected into CEF cells. After 4-5 days, the transfected cells were observed for the appearance of MDV-specific plaque lesions (CPE). After the appearance of plaques, the virus was harvested and frozen in liquid nitrogen. The genomic DNA of the recombinant virus was extracted using a kit, identified by PCR using primers VP2F / R and Fiber2-F / R, and sequenced. The genomic DNA of the parental virus was used as a negative control. The correctly identified recombinant MDV was named rMDV-VP2-Fiber2.

[0040] .

[0041] 1.8 Detection of recombinant virus replication ability The recombinant virus rMDV-VP2-Fiber2 and the parental virus rMSΔmeq strain were inoculated on CEF in 6-well plates at a dose of 100 plaque-forming units (PFU), and 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 growth curve was drawn to analyze the in vitro replication characteristics of the recombinant virus rMDV-VP2-Fiber2 and the parental virus in CEF.

[0042] 1.9 Identification of recombinant virus gene expression 100 PFU of recombinant virus rMDV-VP2-Fiber2 and parental virus rMSΔmeq were inoculated into CEFs, and after 3-4 days of culture, CPE appeared. The cells were fixed with anhydrous ethanol, and the expression of Fiber2 and VP2 proteins was identified by indirect immunofluorescence assay (IFA) using Fiber2 protein polyclonal antibody as primary antibody, FITC-labeled goat anti-mouse IgG (1:200) as secondary antibody, VP2 protein MAb (1:400) as primary antibody, and FITC-labeled anti-mouse IgG (1:200) as secondary antibody. CEFs inoculated with parental virus were used as negative control in IFA.

[0043] 1.10 FAdV-4 challenge protection test Twenty one-day-old SPF chickens were divided into two groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus rMDV-VP2-Fiber2 at a dose of 2000 PFU / chicken through the neck; Group 2 was not inoculated and served as the challenge control group. 28 days after immunization, the rMDV-VP2-Fiber2 immunization group and the challenge control group were inoculated with 2000 PFU / chicken of the FAdV-4 strong strain HLJFAd15 via pectoral intramuscular injection, and the blank control group was not challenged and served as a healthy control. After the challenge, the chickens were observed for 7 days, and the survival of the chickens in each group was counted.

[0044] 1.11 IBDV challenge protection test Twenty one-day-old SPF chickens were divided into two groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus rMDV-VP2-Fiber2 at a dose of 2000 PFU / chicken; Group 2 was not inoculated as the challenge control group. 28 days after immunization, the chickens in the immunization group and the challenge control group were challenged with IBDV super-virulent virus (HLJ0504 strain) at a dose of 10 CLD50. After the challenge, the chickens were observed for 7 days, and the survival of the chickens in each group was counted.

[0045] 1.12 Data Analysis The antibody level, replication titer and other detection data were statistically analyzed using Prism 10 software, and the t-test method was used for significant difference analysis, with P<0.05 representing a significant difference.

[0046] 2. Results 2.1 Construction and identification of recombinant cosmids expressing target genes The VP2 gene and its expression cassette were obtained by amplification technology, and then the gene expression cassettes were inserted into the rMS-ΔMeq recombinant cosmid pMS-4 using Red / ET recombination technology to successfully construct the pMS-VP2 cosmid. The newly constructed plasmid was amplified by PCR and sequenced using primers VP2F / R. The results showed that the length of the PCR product was 1359 bp ( tú1 ). The sequencing results confirmed that the PCR product contained the VP2 gene sequence, while the VP2 gene sequence was not detected in the negative control sample, and the PCR result was negative, which confirmed the successful construction of the pMS-VP2 cosmid. Similarly, the Fiber2 gene and its expression cassette were amplified, and the Fiber2 gene expression cassette was cloned into the rMS-ΔMeq recombinant cosmid pMS-3 using Red / ET recombination technology, and the pMS-Fiber2 cosmid was successfully constructed. After PCR amplification and sequencing identification, it was confirmed that the construction of the pMS-Fiber2 cosmid was also successful ( tú1 ).

[0047] 2.2 Rescue of recombinant viruses and PCR identification The recombinant cosmids pMS-VP2 and pMS-Fiber2 were transfected into CEF cells together with three other recombinant cosmids pMS-1, pMS-2, and pMS-5 containing rMS-ΔMeq genome fragments. MDV plaque lesions were observed 4 to 5 days after transfection, and the recombinant Marek's disease virus rMDV-VP2-Fiber2 ( tú2 ). Subsequently, the genomic DNA of the recombinant virus was extracted and PCR identification and sequencing analysis were performed. The results showed that the length of the PCR product was consistent with the expectation ( tú3), the sequencing results also confirmed that the gene fragment CMV-VP2 containing the CMV promoter sequence, IBDV VP2 gene and terminator sequence was inserted into the US2 gene of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain, and replaced the nucleotide sequence of 156501-157116 of the rMSΔMeq strain genome sequence. At the same time, the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, FAdV-4 Fiber2 gene and terminator sequence was inserted into the UL41 gene of the rMSΔMeq strain, and replaced the nucleotide sequence of 96927-97106 of the rMSΔMeq strain genome sequence. The insertion sequence was consistent with expectations. At the same time, the genomic DNA amplification result of the parental virus rMS-ΔMeq strain was negative. These results jointly confirmed that the VP2 gene and Fiber2 gene had been correctly inserted into the rMS-ΔMeq genome and could exist stably, indicating that the construction of the recombinant virus rMDV-VP2-Fiber2 was correct.

[0048] 2.3 Expression of target genes VP2 and Fiber2 After the recombinant Marek's disease virus rMDV-VP2-Fiber2 and the parental virus rMS-Δmeq strain were inoculated into CEF cells, cell samples were collected 120 hours later and the expression of the target genes VP2 and Fiber2 was detected using indirect immunofluorescence assay (IFA). The experimental results showed that cells infected with rMDV-VP2-Fiber2 showed a positive reaction to VP2 monoclonal antibody and Fiber2 polyclonal antibody, while no fluorescence signal was observed in cells infected with the parental MDV virus and uninfected cells ( tú4 ). These results confirmed that the recombinant virus rMDV-VP2-Fiber2 could successfully express the target genes VP2 and Fiber2 simultaneously.

[0049] 2.4 Identification of the in vitro replication kinetics of recombinant viruses The recombinant virus rMDV-VP2-Fiber2 and the parental virus rMSΔmeq were inoculated with 100 PFU into the secondary CEF cells cultured in 6-well plates, and the cells inoculated with the virus were collected every 24 hours. The virus titers collected at different time points were measured and growth curves were drawn. The results showed that both the recombinant virus rMDV-VP2-Fiber2 and the parental virus rMSΔmeq reached the peak of replication titer 120 hours after infection, and at each time point after infection of CEF cells, the titer of the recombinant virus was not significantly different from that of the parental virus (p>0.05) ( tú5 ), which indicated that the replication properties of rMDV-VP2-Fiber2 in CEF cells were similar to those of the parental virus.

[0050] 2.5 Evaluation of the immune protection of rMDV-VP2-Fiber2 against FAdV-4 The recombinant Marek's disease virus rMDV-VP2-Fiber2 was inoculated into one-day-old SPF chickens at a dose of 2000 PFU / chicken, and FAdV-4 was challenged 4 weeks after immunization. The mortality rate was counted 7 days after the challenge, and the results showed that 8 chickens in the experimental group vaccinated with rMDV-VP2-Fiber2 survived the challenge with FAdV-4 (HLJFAd15), and the surviving chickens showed no obvious symptoms ( tú6 ). In contrast, the immunized group vaccinated with the parental virus rMS-Δmeq all showed obvious clinical symptoms after FAdV-4 challenge, and 8 out of 10 chickens died. These results show that the protection rate of chickens vaccinated with the recombinant Marek's disease virus rMDV-VP2-Fiber2 against FAdV-4 challenge reached 80%.

[0051] 2.6 Evaluation of rMDV-VP2-Fiber2 immune protection against IBDV The recombinant Marek's disease virus rMDV-VP2-Fiber2 was inoculated into 1-day-old SPF chickens at a dose of 2000 PFU / chicken, and the chickens were challenged with the IBDV super-virulent strain (HLJ0504) 4 weeks after immunization. The mortality rate was counted 7 days after the challenge, and the results showed that the chickens in the test group vaccinated with rMDV-VP2-Fiber2 survived the challenge with the IBDV super-virulent strain completely, and the surviving chickens showed no obvious symptoms ( tú7 ). In contrast, the group vaccinated with the parental virus rMS-Δmeq showed obvious clinical symptoms after the IBDV super-virulent strain challenge, and 10 out of 10 chickens died. These results show that the protection rate of chickens vaccinated with the recombinant Marek's disease virus rMDV-VP2-Fiber2 against the IBDV super-virulent strain challenge reached 100%.

Claims

1. A recombinant Marek's disease virus expressing IBDV VP2 and FAdV-4 Fiber2 genes, characterized in that: The gene fragment CMV-VP2 containing the CMV promoter sequence, the Infection Bursal Disease Virus (IBDV) VP2 gene and the terminator sequence is inserted into the Marek's disease virus Meq Gene-deleted vaccine strain rMSΔMeq US2 The nucleotide sequence at positions 156501-157116 of the rMSΔMeq strain genome sequence was replaced inside the gene. At the same time, the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, the serotype 4 avian adenovirus (FAdV-4) Fiber2 gene and the terminator sequence was inserted into the Marek's disease virus Meq Gene-deficient vaccine strains UL41 The gene was inserted into the nucleotide sequence of position 96927-97106 of the rMSΔMeq strain genome sequence, thereby obtaining a recombinant Marek's disease virus strain that simultaneously expresses the IBDVVP2 gene and FAdV-4 Fiber2; Wherein, the Marek's disease virus Meq The gene-deficient vaccine strain rMSΔMeq strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and its microbiological deposit number is: CGMCC No.4612; The VP2 gene is from the infectious bursal disease virus HLJ0504 strain, and the Fiber2 gene is from the serotype 4 avian adenovirus virulent strain HLJFAd15 strain, whose GenBank sequence number is KU991797; Among them, the nucleotide sequence of the gene fragment CMV-VP2 is shown in SEQ ID NO.15, and the nucleotide sequence of the gene fragment SV40-Fiber2 is shown in SEQ ID NO.

14.

2. A method for constructing the recombinant Marek's disease virus according to claim 1, characterized in that: The following steps are involved: (1) Marek's disease virus Meq Construction of the reverse genetics operating system for the gene-deficient vaccine strain rMSΔMeq: The reverse genetics operating system comprises 5 cosmids, which are Marek's disease virus Meq The gene fragment of the gene-deficient vaccine strain rMSΔMeq was cloned into pCC1Fos. The five cosmids contain Marek's disease virus Meq The nucleotide sequences of the genome of the gene-deficient vaccine strain rMSΔMeq strain at positions 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526 are named pMS-1, pMS-2, pMS-3, pMS-4 and pMS-5, respectively, wherein pMS-3 comprises the UL41 gene of the rMSΔMeq strain, and pMS-4 comprises the US2 gene of the rMSΔMeq strain; the Marek's disease virus Meq The genome sequence GenBank accession number of the gene-deleted vaccine strain rMSΔMeq is JQ314003.1; (2) Construction and identification of recombinant cosmids pMS-VP2 and pMS-Fiber2 expressing target genes: pMS-3 cosmid and pMS-4 cosmid were transfected into competent cells containing pRed E / T expression plasmid to prepare corresponding competent cells; rpsl-neo expression cassette DNA was used as template, primers UL41-rpsl F / R and US2-rpsl F / R were used to amplify by PCR to obtain rpsl-neo gene fragments containing homologous arms at both ends of the UL41 insertion site and homologous arms at both ends of the US2 insertion site, respectively, which were purified and transfected into competent cells containing pMS-3 and pMS-4 to prepare competent cells; IBDV VP2 gene expression plasmid was used as template, primers US2-CMV F / R were used to amplify by PCR to obtain CMV-VP2 gene fragments containing homologous arms at both ends of the US2 insertion site, respectively, which were purified and electroporated into rpsl-neo competent cells containing homologous arms at both ends of the US2 insertion site; FAdV-4 Fiber2 gene expression plasmid was used as template, primers UL41-SV40 F / R was amplified by PCR to obtain the Fiber2 gene fragment containing the homologous arms at both ends of the UL41 insertion site, which was purified and electroporated into the competent cells of rpsl-neo containing the homologous arms at both ends of the UL41 insertion site; positive colonies were picked and expanded for culture and then the cosmids were extracted, and the obtained cosmids were named pMS-VP2 and pMS-Fiber2 respectively; the names and sequences of the primers required for construction are as follows: ； The IBDV VP2 gene expression plasmid is obtained by amplifying the VP2 gene using the genome of the IBDV HLJ-0504 strain as a template and then cloning it into the CMV expression box of the vector pEGFP-N1; the FAdV-4 Fiber2 gene expression plasmid is obtained by amplifying the Fiber2 gene using the genome DNA of the FAdV-4 virulent strain HLJFAd15 as a template and then cloning it into the BstB Ⅰ and Sma Ⅰ sites of the pCDNA3.1 vector to obtain the plasmid pSV40-Fiber2; The nucleotide sequence of the gene fragment CMV-VP2 in the IBDV VP2 gene expression plasmid containing the CMV promoter sequence, the IBDV VP2 gene and the terminator sequence is shown in SEQ ID NO.15; the nucleotide sequence of the gene fragment SV40-Fiber2 in the FAdV-4 Fiber2 gene expression plasmid containing the SV40 promoter, the FAdV-4 Fiber2 gene and the terminator sequence is shown in SEQ ID NO.14; (3) Rescue of recombinant Marek's disease virus rMDV-VP2-Fiber2: The recombinant cosmids pMS-VP2 and pMS-Fiber2 and the other three parental cosmids pMS1, pMS2 and pMS5 cloned with rMSΔmeq genomic fragments were extracted using a plasmid extraction kit. The five cosmids were purified by the calcium phosphate method. Not The linearized cosmids were co-transfected into CEF cells. After 4-5 days, the transfected cells were observed for the appearance of MDV-specific plaques. After the appearance of plaques, the viruses were harvested and frozen in liquid nitrogen. The genomic DNA of the recombinant virus was extracted and identified by PCR and sequencing. The correctly identified recombinant MDV was named rMDV-VP2-Fiber2.

3. Use of the recombinant Marek's disease virus according to claim 1 in the preparation of a medicament for preventing FAdV-4 and / or IBDV infection.

4. The use according to claim 3, characterized in that The FAdV-4 is a strong strain of FAdV-4; and the IBDV is a super strong strain of IBDV.

5. The use according to claim 3, characterized in that The medicine is a recombinant Marek's disease virus live vector vaccine expressing IBDV VP2 gene and FAdV-4Fiber2 gene.

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