Recombinant Marek's disease virus for expressing serum 4 type fowl adenovirus Fiber2 gene 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 a serum 4 type fowl adenovirus Fiber2 gene 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 obtained by inserting a gene segment SV40-Fiber2 containing an SV40 promoter sequence, an FAdV-4 Fiber2 gene and a terminator sequence into a UL41 gene of a Marek's disease virus Meq gene deleted vaccine strain. A challenge protection test result shows that after chicken is immunized by the recombinant virus rMDV-4 Fiber2, good protection can be provided for FAdV-4 and MDV challenge, and 80% protection and 100% protection are respectively provided for death caused by FAdV-4 virulent strain and MDV virulent attack. The construction of the recombinant virus rMDV-Fiber2 provides a technical means for the development of HHS recombinant MDV live vector vaccines, and is of great significance to the prevention and control of HHS.

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 a serum type 4 avian adenovirus Fiber2 gene and a construction method and application thereof. The present invention belongs to the field of biotechnology. Background Art

[0002] Hydropericardium hepatitis syndrome (HHS) is an acute, highly contagious and immunosuppressive infectious disease of chickens caused by serotype 4 fowl adenovirus (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 to have ruffled feathers and depression, and autopsy can show 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. Studies have shown that using Fiber2 as a subunit vaccine to immunize animals can provide 100% protection against FAdV-4 attacks. Currently available FAdV-4 vaccines are mostly multi-component and multivalent vaccines. For example, there are inactivated two-component vaccines containing FAdV-4 and NDV or EDSV abroad. There are inactivated three-component vaccines composed of NDV, AIV and FAdV-4 in China. However, there are certain limitations in the use and development of inactivated vaccines, such as the need to operate multiple pathogens and multiple immunizations, which increases the cost of vaccine production and application. Recombinant vector vaccines can achieve the effect of multiple protections with one shot, and the laboratory construction is relatively simple, which can make up for the shortcomings of existing vaccines.

[0003] Marek's disease (MD) is a contagious tumor disease of chickens caused by Marek's disease virus (MDV). Due to the high infectivity and tumorigenicity of MDV, MD has become one of the most harmful infectious diseases to the global poultry industry. MDV is divided into three serotypes. The main wild strains that can cause disease are serotype 1 (MDV 1), serotype 2 (MDV 2) includes some non-pathogenic strains clinically isolated from healthy chickens, and serotype 3 (MDV3) is represented by herpesvirus of turkeys (HVT). The Meq gene-deficient vaccine strain rMSΔMeq is obtained by deleting the first half of the 468bp base sequence of the main tumorigenic gene Meq gene based on the parent strain MDV MS strain. Its genome is large and has many replication-non-essential regions for the insertion of exogenous genes. It is an ideal vector for constructing recombinant live vaccines. The present invention uses rMSΔMeq as a skeleton, a multi-fragment cosmid rescue system and Red / ET recombination technology, inserts the FAdV-4 Fiber2 gene expression cassette into the rMSΔMeq genome, constructs a recombinant virus rMDV-Fiber2 expressing the Fiber2 gene, analyzes its in vitro biological characteristics, and preliminarily evaluates its protection effect against FAdV-4 attack through a virus protection test, providing a technical means for the development of HHS recombinant MDV live vector vaccine. Summary of the invention

[0004] The object of the present invention is to provide a recombinant Marek's disease virus expressing the Fiber2 gene of serotype 4 fowl adenovirus, which is safe to use, has a simple immunization procedure and a long immunization duration, and a method for constructing the same.

[0005] In order to achieve the above object, the present invention adopts the following technical means: The present invention provides a recombinant Marek's disease virus expressing the Fiber2 gene of serotype 4 fowl adenovirus, wherein the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, the Fiber2 gene of serotype 4 fowl adenovirus and the terminator sequence is inserted into the Marek's disease virus Meq Gene-deficient vaccine strains UL41 Acquired within the gene.

[0006] 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.

[0007] Preferably, the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, FAdV-4 Fiber2 gene and terminator sequence is inserted into the rMSΔMeq strain. UL41 The nucleotide sequence at positions 96927-97106 of the rMSΔMeq strain genome sequence was replaced within the gene to obtain a recombinant Marek's disease virus strain expressing the FAdV-4 Fiber2 gene.

[0008] Preferably, the Fiber2 gene is from the HLJFAd15 strain, a virulent avian adenovirus serotype 4 strain, and its GenBank sequence number is KU991797.

[0009] Among them, preferably, the nucleotide sequence of the gene fragment SV40-Fiber2 is shown in SEQ ID NO.14.

[0010] 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; the Marek's disease virus Meq The genome sequence GenBank accession number of the gene-deleted vaccine strain rMSΔMeq is JQ314003.1; (2) Construction of Fiber2 expression plasmid pSV40-Fiber2 First, the Fiber2 gene fragment was amplified from the FAdV-4 virulent strain HLJFAd15 using PCR primers Fiber2 F / R, and then the Fiber2 fragment was ligated to the pCDNA3.1 vector. BstB Ⅰ and SmaI site, and obtained plasmid pSV40-Fiber2, and 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; the primer sequence is shown below: ; (3) Construction of recombinant cosmid expressing Fiber2 gene The recombinase plasmid pRed E / T was electrotransformed into competent cells DH10B, and positive colonies were picked to prepare pRed E / T expression competent cells. Then, the pMS-3 cosmid was electrotransformed into competent cells containing pRed E / T expression plasmids, and positive colonies were picked to prepare competent cells. Using rpsl-neo as a template, primers UL41-rpsl F / R were used to amplify the rpsl-neo gene fragment containing homologous arms at both ends of the UL41 insertion site by PCR, which was purified and electrotransformed into competent cells containing pMS-3, and positive colonies were picked to prepare competent cells. Using FAdV-4 Fiber2 gene expression plasmid as a template, primers UL41-SV40 F / R were used to amplify the Fiber2 gene fragment containing homologous arms at both ends of the UL41 insertion site, which were purified and electrotransformed into competent cells containing rpsl-neo, positive colonies were picked to expand and culture, and cosmids were extracted, and the obtained cosmids were named pMS-Fiber2. The primer sequences are as follows: ; (4) Rescue and identification of recombinant MDV expressing Fiber2 The recombinant cosmid pMS-Fiber2 and the other four parental cosmids pMS-1, pMS-2, pMS-4, and pMS-5 cloned with the rMSΔMeq genomic fragment were extracted using a plasmid extraction kit. The five parental cosmids pMS-1, pMS-2, pMS-4, and pMS-5 were cloned with the calcium phosphate method. Not The linearized cosmids were co-transfected into CEF cells with enzyme I. After 4-5 days, the transfected cells were observed for the appearance of MDV-specific plaque lesions. After the appearance of plaques, the virus was harvested and frozen in liquid nitrogen. After PCR identification and sequencing, a recombinant Marek's disease virus expressing the Fiber2 gene of serotype 4 fowl adenovirus was obtained.

[0011] Among them, preferably, the FAdV-4 Fiber2 gene expression plasmid is prepared by using the genomic DNA of the FAdV-4 virulent strain HLJFAd15 as a template to amplify the Fiber2 gene and then clone 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 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.

[0012] 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 MDV infection.

[0013] Among them, preferably, the FAdV-4 is a strong strain of FAdV-4; and the MDV is a strong strain of MDV.

[0014] Among them, preferably, the drug is a recombinant Marek's disease virus live vector vaccine expressing the FAdV-4 Fiber2 gene.

[0015] Compared with the prior art, the beneficial effects of the present invention are: The present invention utilizes the recombinant cloning technology to insert the gene fragment SV40-Fiber2 containing the serotype 4 avian adenovirus Fiber2 gene and the SV40 promoter sequence into the UL41 gene of the Marek's disease virus gene-deficient vaccine strain (rMS-ΔMeq), and constructs a recombinant cosmid pMS-Fiber2 containing the SV40-Fiber2 expression frame. pMS-Fiber2 and other four overlapping cosmids covering the entire MDV genome are co-transfected into chicken embryo fibroblasts (CEF) to rescue the recombinant virus rMDV-Fiber2 expressing the serotype 4 avian adenovirus Fiber2 gene. The research results show that rMDV-Fiber2 can stably express the Fiber2 protein, and the replication ability of rMDV-Fiber2 in CEF is not significantly different from that of the parent virus rMSΔMeq. The results of the challenge protection test showed that the recombinant virus rMDV-Fiber2 can provide good protection against FAdV-4 and MDV attacks after immunizing chickens, providing 80% protection against death caused by FAdV-4 strong strains, and 100% immune protection against death caused by MDV strong virus attacks. The construction of the recombinant virus rMDV-Fiber2 provides a technical means for the development of HHS recombinant MDV live vector vaccines, which is of great significance for the prevention and control of HHS. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of the construction of recombinant cosmids; FIG2 is a diagram showing the results of PCR identification of the recombinant cosmid pMS-Fiber2; Wherein, M: DNA molecular mass standard; 1: recombinant cosmid pMS-Fiber2; 2: negative control (pMS-3); FIG3 is a diagram showing the microscopic observation results of the recombinant virus rMDV-Fiber2: Among them, (a) uninfected CEF under optical microscope; (b) F3 generation recombinant virus CPE under optical microscope; (c) MDV virus particles under electron microscope; Fig. 4 is a diagram showing the results of PCR identification of the recombinant virus rMDV-Fiber2; Wherein, M: DNA molecular mass standard; 1: recombinant virus rMDV-Fiber2; 2: negative control rMSΔMeq; Fig. 5 is a diagram showing the results of IFA identification of the recombinant virus rMDV-Fiber2; FIG6 is a diagram showing the results of WB identification of the recombinant virus rMDV-Fiber2; Among them, 1: rMDV-Fiber2; 2: rMSΔMeq; 3: blank CEF cells; FIG7 is a graph showing a comparison of the replication kinetics of the recombinant virus rMDV-Fiber2 and the parental virus rMSΔMeq; FIG8 is a graph showing the protection results of SPF chickens immunized with the recombinant virus rMDV-Fiber2 against HLJFAd15; Figure 9 shows the protection results of SPF chickens immunized with the recombinant virus rMDV-Fiber2 against Md5. DETAILED DESCRIPTION

[0017] The present invention is further described below by way of examples, the purpose of which is only to provide a better understanding of the research content of the present invention but not to limit the protection scope of the present invention. Except for special examples, the following examples are all conventional experimental methods and operating steps in the art.

[0018] 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 Meq Fragments 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).

[0019] 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.

[0020] 4) Construction of transfer vector plasmid: Using genomic DNA of MDV MS strain as template, the transfer vector plasmid was obtained by PCR method.Meq The 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.

[0021] 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 2mL 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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. 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.

[0027] 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.

[0028] Example 2 Construction and Identification of Recombinant Marek's Disease Virus Expressing the Fiber2 Gene of Serotype 4 Avian Adenovirus 1. Materials and methods: 1.1 Plasmids, strains, and viruses The pCDNA3.1 vector was preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the E. coli EPI300 strain was purchased from EPICENTRE; the FAdV-4 virulent strain HLJFAd15 (GenBank sequence number is KU991797) and the MDV virulent strain Md5 (GenBank Acc. No.: AF243438.1) were preserved and provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Chicken embryo fibroblasts (CEF) were prepared from 8-10 day old SPF chicken embryos.

[0029] 1.2 Main Reagents Conventional PCR reaction enzymes, restriction endonucleases, and ligases were purchased from TaKaRa; calcium phosphate transfection reagents were purchased from Invitrogen; plasmid extraction kits were purchased from QIAGEN; body fluid viral DNA / RNA minipreparation kits were purchased from Suzhou Youyi Landi Biotechnology Co., Ltd.; cell lysate was purchased from Bio-Time; cell culture medium and fetal bovine serum were purchased from Gibco; mouse anti-rabbit IgG-FITC and IRDye-800-labeled goat anti-mouse IgG were purchased from Licor; β-actin monoclonal antibody was purchased from Sigma; gI monoclonal antibody and Fiber2 polyclonal antibody were prepared and preserved by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0030] 1.3 Construction of Fiber2 expression plasmid pSV40-Fiber2 First, the Fiber2 gene fragment was amplified from the FAdV-4 virulent strain HLJFAd15 using PCR primers Fiber2 F / R (F: 5¢-GACAGGATGAGGATCGTTTCGCGCCACCATGCTCCGGGCCCCTAAA-3¢ (SEQ ID NO.12); R: 5¢-TCGGTCATTTCGAACCCCAGAGTCCCGCTTACGGGAGGGAGGCCGC-3¢ (SEQ ID NO.13)), and then the Fiber2 fragment was ligated to 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.

[0031] 1.4 Construction of a reverse genetics system for the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq The reverse genetic operating system comprises five cosmids containing the nucleotide sequences of the genome of the rMSΔMeq strain of the Meq gene-deficient Marek's disease virus, respectively, at positions 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526. Each cosmid is obtained by cloning the gene fragment of the corresponding rMSΔMeq strain of the Meq gene-deficient Marek's disease virus into pCC1Fos using the Copy Control Fosmidlibrary production kit, and they 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 the GenBank accession number of the genome sequence of the rMSΔMeq strain of the Meq gene-deficient Marek's disease virus is JQ314003.1.

[0032] The GenBank accession number of the genome sequence of the Marek's disease virus Meq gene deleted vaccine strain rMSΔMeq strain is JQ314003.1.

[0033] 1.5 Construction of recombinant cosmid expressing Fiber2 gene The recombinant cosmid was constructed using a two-step Red / ET recombination method: the recombinase plasmid pRed E / T was electroporated into the competent cell DH10B, and the positive colonies were picked to prepare the pRed E / T expression electroporated competent cells. Then, the pMS-3 cosmid was electroporated into the competent cells (containing the pRed E / T expression plasmid), and the positive colonies were picked to prepare the competent cells. Using rpsl-neo as a template, primers UL41-rpsl F / R (F: 5¢-ATCTGTGTCCGAAGACAATACATATGCGACGGCCTGGTGATGATGGCGGG-3¢ (SEQ ID NO.15); R: 5¢-GATGAATGTGGAAGTACGGATGTTGGAGAGTCAGAAGAACTCGTCAAGAA-3¢ (SEQ ID NO.16)) were used for PCR amplification to obtain the rpsl-neo gene fragment containing the homologous arms at both ends of the UL41 insertion site. After purification, it was electroporated into competent cells containing pMS-3, and the positive colonies were picked to prepare the competent cells. Using the expression plasmid pSV40-Fiber2 as a template, primers UL41-SV40 F / R (F: 5¢-ATCTGTGTCCGAAGACAATACATATGCGACGTGTGTCAGTTAGGGTGTGG-3¢ (SEQ ID NO.17); R: 5¢-GATGAATGTGGAAGTACGGATGTTGGAGAGTAAGATACATTGATGAGTTT-3¢ (SEQ ID NO.18)) were used to amplify the Fiber2 gene fragment containing the homologous arms at both ends of the UL41 insertion site. The fragments were purified and electroporated into competent cells containing rpsl-neo. Positive colonies were picked and expanded for culture, and then the cosmids were extracted. The obtained cosmid was named pMS-Fiber2.

[0034] 1.6 Rescue and identification of recombinant MDV expressing Fiber2 The recombinant cosmid pMS-Fiber2 and the other four parental cosmids pMS-1, pMS-2, pMS-4, and pMS-5 cloned with the rMSΔMeq genomic fragment were extracted using a plasmid extraction kit. The five parental cosmids pMS-1, pMS-2, pMS-4, and pMS-5 were cloned with the calcium phosphate method. NotI was digested and linearized with cosmids and 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 and identified by PCR using primers WD-JD F / R (F: 5¢-GCTGTCTCATCACGTCCTC-3¢ (SEQ ID NO.19); R: 5¢-AAAAACTGCGAGGGTCATCA-3¢ (SEQID NO.20)). The genomic DNA of the parental virus rMSΔMeq strain was used as a negative control.

[0035] 1.7 Detection of the replication ability of recombinant viruses The recombinant virus rMDV-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-Fiber2 and the parental virus rMSΔMeq strain in CEF.

[0036] 1.8 Identification of recombinant virus gene expression 100 PFU of recombinant virus rMDV-Fiber2 and parental virus rMSΔMeq were inoculated into CEF, and after CPE appeared for 3-4 days, the cells were fixed with anhydrous ethanol. Subsequently, FAdV-4 Fiber2 mouse polyclonal antibody (1:500) was used as primary antibody, FITC-labeled goat anti-mouse IgG antibody (1:200) was used as secondary antibody, and CEF samples inoculated with rMSΔmeq were used as negative control to detect whether Fiber2 protein was expressed by indirect immunofluorescence assay (IFA).

[0037] 100 PFU of recombinant virus rMDV-Fiber2 and parental virus rMSΔMeq were inoculated into CEFs, and after 3-4 days of culture, CPE appeared. The infected cells were lysed with cell lysis buffer, and the supernatant of the lysed cells was subjected to SDS-PAGE gel electrophoresis and transferred to NC membrane. After the transfer, FAdV-4 Fiber2 mouse polyclonal antibody (1:500) was used as the primary antibody, and IRDye-800 labeled goat anti-mouse IgG (1:10000) was used as the secondary antibody to identify whether Fiber2 protein was expressed by Western-blot; at the same time, CEFs inoculated with rMSΔmeq were used as negative controls, and CEFs not inoculated with the virus were used as blank controls. At the same time, parallel groups were set up with mouse monoclonal antibodies against the internal reference protein β-actin as primary antibodies and mouse monoclonal antibodies against MDV gI as primary antibodies.

[0038] 1.9 FAdV-4 challenge protection test Thirty one-day-old SPF chickens were divided into three groups, with 10 in each group. Group 2 (rMDV-Fiber2 immune challenge group) was subcutaneously inoculated with the recombinant virus rMDV-Fiber2 at a dose of 2000 PFU / chicken; Group 1 (blank control group) and Group 3 (non-immune challenge group) were not inoculated and served as the control group. 28 days after immunization, blood was collected from the sub-wing vein before the challenge and serum was separated. The rMDV-Fiber2 immune challenge group and the non-immune challenge group were inoculated with 2000 PFU / chicken of the FAdV-4 strong strain HLJFAd15 through the pectoral muscle injection route, and the blank control group was not challenged 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.

[0039] 1.10 MDV 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-Fiber2 at a dose of 2000 PFU / chicken; Group 1 was not inoculated as the challenge control group. Seven days after immunization, the chickens in the immunized group and the challenge control group were inoculated with 1000 PFU / chicken of the MDV virulent strain Md5 by intraperitoneal injection. After the challenge, the chickens were observed for 12 weeks and the survival of the chickens in each group was counted.

[0040] 1.11 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.

[0041] 2. Results 2.1 Construction and identification of recombinant cosmid pMS-Fiber2 The amplified Fiber2 gene and expression cassette were used to construct a recombinant cosmid using the Red / ET recombination method, and the Fiber2 gene expression cassette was cloned into the rMSΔMeq recombinant cosmid pMS-3 to construct the pMS-Fiber2 cosmid ( Figure 1 ); The recombinant cosmid was identified by PCR and sequencing using primers Fiber2 F / R. The results showed that the length of the obtained PCR product was about 1.4 kb ( Figure 2 ), sequencing results showed that the PCR product contained the Fiber2 gene, while the negative control sample had no Fiber2 gene insertion sequence, and the PCR identification result was negative, indicating that the recombinant cosmid pMS-Fiber2 was successfully constructed.

[0042] 2.2 Rescue and PCR identification of recombinant virus rMDV-Fiber2 The recombinant cosmid pMS-Fiber2 was co-transfected into CEF with four other recombinant cosmids pMS-1, pMS-2, pMS-4, and pMS-5 cloned with MDV genome fragments. After three generations of blind transfection, typical MDV plaques were observed in CEF under an optical microscope, which were scattered grape-like, while the CEF cells in the blank control group had no MDV plaques; MDV virus-like particles ( Figure 3 ), using the parental virus rMSΔmeq genomic DNA as a control, the genomic DNA of the recombinant virus was extracted and PCR identification and sequencing analysis showed that the length of the amplified product was about 3.2 kb ( Figure 4 ), which is consistent with the expected results. The sequencing results showed that the gene fragment SV40-Fiber2 containing the SV40 promoter sequence, FAdV-4 Fiber2 gene and terminator sequence was correctly inserted into the UL41 gene of the rMSΔMeq genome and replaced the nucleotide sequence at positions 96927-97106 of the rMSΔMeq strain genome sequence. The insertion sequence was consistent with expectations. The above results show that the Fiber2 gene was correctly inserted into the rMSΔmeq genome and can exist stably, and the recombinant virus rMDV-Fiber2 was correctly constructed.

[0043] 2.3 Expression and identification of recombinant virus rMDV-Fiber2 protein The recombinant virus rMDV-Fiber2 and the parental virus rMSΔMeq were inoculated into CEF, and CPE appeared after 3-4 days of culture. The IFA results showed that the rMDV-Fiber2 infected cells could specifically react with the Fiber2 polyclonal antibody, showing clear green fluorescence under the fluorescence microscope. In contrast, no fluorescence was observed in the parental virus rMSΔMeq infection group ( Figure 5), confirming that the Fiber2 protein was successfully expressed in the recombinant virus. Western blot results showed that the expression of Fiber2 protein could be detected in cells infected with the recombinant virus rMDV-Fiber2. The protein size was 69 ku ( Figure 6 ), while the expression of this protein was not detected in the parental virus rMSΔmeq and blank control group cells, which was in line with experimental expectations. The internal reference protein β-actin was stably expressed in all groups, confirming that the operation and sample processing were correct; the expression of MDV gI protein in the recombinant virus and the parental virus further verified the success of viral infection. The above IFA and Western-blot results show that the recombinant virus rMDV-Fiber2 can specifically express the Fiber2 protein in infected cells.

[0044] 2.4 Identification of in vitro replication kinetics of the recombinant virus rMDV-Fiber2 The recombinant virus rMDV-Fiber2 and the parent virus rMSΔMeq were inoculated with 100 PFU into the secondary CEF cells cultured in 6-well plates, and the virus-infected cells in the wells were digested and collected every 24 hours. The titers of the virus samples collected at each time point were measured, and the virus growth curve was drawn accordingly. The results showed that at 120 hours after infection, the replication titers of rMDV-Fiber2 and rMSΔmeq reached a peak ( Figure 7 ), titer analysis at different time points showed that there was no statistical difference in the proliferation of the recombinant virus and the parental virus in CEF cells (p>0.05). The above results indicate that inserting the Fiber2 gene expression cassette into the UL41 site of the rMSΔMeq gene does not affect the replication ability of the recombinant virus on CEF.

[0045] 2.5 Evaluation of the immune protection of the recombinant virus rMDV-Fiber2 against FAdV-4 The recombinant virus rMDV-Fiber2 was inoculated at a dose of 2000 PFU / chicken into one-day-old SPF chickens in the rMDV-Fiber2 immune challenge group. The rMDV-Fiber2 immune challenge group and the non-immune challenge group were challenged 28 days after immunization. The results showed that 8 out of 10 chickens in the rMDV-Fiber2 immune challenge group survived after being challenged with FAdV-4, while the non-immune challenge group showed obvious clinical symptoms after challenge, and 10 out of 10 chickens died ( Figure 8 ). The above results show that the recombinant virus rMDV-Fiber2 can provide 80% immune protection against FAdV-4 virulent attack after immunization.

[0046] 2.6 Evaluation of the immune protection of the recombinant virus rMDV-Fiber2 against MDV The recombinant virus rMDV-Fiber2 was inoculated into one-day-old SPF chickens at a dose of 2000 PFU / chicken. Seven days after immunization, the rMDV-Fiber2 immune group and the challenge control group were challenged with the MDV virulent strain Md5, and the chickens were observed for 12 weeks. The results showed that all 10 chickens immunized with the recombinant virus rMDV-Fiber2 survived the challenge with Md5, while the challenge control group showed obvious clinical symptoms after the challenge with Md5, and 9 out of 10 chickens died ( Figure 9 ). The above results show that the recombinant virus rMDV-Fiber2 can provide 100% immune protection against MDV virulent attack after immunization.

Claims

1. A recombinant Marek's disease virus (MDV) expressing the Fiber2 gene of serotype 4 fowl adenovirus (FAdV-4), characterized in that: The gene fragment SV40-Fiber2 containing the SV40 promoter sequence, the serotype 4 fowl adenovirus fiber 2 gene and the terminator sequence is inserted into the Marek's disease virus Meq Gene-deleted vaccine strain rMSΔMeq UL41 gene, and replace the nucleotide sequence at positions 96927-97106 of the rMSΔMeq strain genome sequence to obtain a recombinant Marek's disease virus strain expressing the FAdV-4 Fiber2 gene; 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; Among them, 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; the Marek's disease virus Meq The genome sequence GenBank accession number of the gene-deleted vaccine strain rMSΔMeq is JQ314003.1; (2) Construction of Fiber2 expression plasmid pSV40-Fiber2: First, the Fiber2 gene fragment was amplified from the FAdV-4 virulent strain HLJFAd15 using PCR primers Fiber2 F / R, and then the Fiber2 fragment was ligated to the pCDNA3.1 vector. B 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; the primer sequences are shown below: ； (3) Construction of recombinant cosmid expressing Fiber2 gene: The recombinase plasmid pRed E / T was electrotransformed into competent cells DH10B, and positive colonies were picked to prepare pRed E / T expression competent cells. Then, the pMS-3 cosmid was electrotransformed into competent cells containing pRed E / T expression plasmids, and positive colonies were picked to prepare competent cells. Using rpsl-neo as a template, primers UL41-rpsl F / R were used to amplify the rpsl-neo gene fragment containing homologous arms at both ends of the UL41 insertion site by PCR, which was purified and electrotransformed into competent cells containing pMS-3, and positive colonies were picked to prepare competent cells. Using FAdV-4 Fiber2 gene expression plasmid as a template, primers UL41-SV40 F / R were used to amplify the Fiber2 gene fragment containing homologous arms at both ends of the UL41 insertion site, which were purified and electrotransformed into competent cells containing rpsl-neo, positive colonies were picked to expand and culture, and the cosmids were extracted, and the obtained cosmids were named pMS-Fiber2. The primer sequences are as follows: ； The FAdV-4 Fiber2 gene expression plasmid is prepared by using the genomic DNA of the FAdV-4 virulent strain HLJFAd15 as a template to amplify the Fiber2 gene and then clone 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 SV40-Fiber2 containing the SV40 promoter, the FAdV-4 Fiber2 gene and the terminator sequence in the FAdV-4 Fiber2 gene expression plasmid is shown in SEQ ID NO.14; (4) Rescue and identification of recombinant MDV expressing Fiber2: The recombinant cosmid pMS-Fiber2 and the other four parental cosmids pMS-1, pMS-2, pMS-4, and pMS-5 cloned with the rMSΔMeq genomic fragment were extracted using a plasmid extraction kit. The five parental cosmids pMS-1, pMS-2, pMS-4, and pMS-5 were cloned with the calcium phosphate method. Not The linearized cosmids were co-transfected into CEF cells with enzyme I. After 4-5 days, the transfected cells were observed for the appearance of MDV-specific plaque lesions. After the appearance of plaques, the virus was harvested and frozen in liquid nitrogen. After PCR identification and sequencing, a recombinant Marek's disease virus expressing the Fiber2 gene of serotype 4 fowl adenovirus was obtained.

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 MDV infection.

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

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

Images