Marek's disease virus gene editing deletion vaccine strain SQ01Δmeq and construction and application thereof

Abstract

The application provides a Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Delta meq and construction and application. The SQ01Delta meq vaccine candidate strain has a preservation number of CGMCC NO.45864. The parent strain of the SQ01Delta meq vaccine candidate strain is MDV super strong Chinese mutant HNSQ01. After in vitro passage of the parent strain HNSQ01, the CRISPR / Cas9 gene editing technology is used to delete a 925bp fragment on each of the two meq alleles in the viral genome. Through PCR identification, gene cloning sequencing, RT-qPCR analysis, IFA identification and SPF chicken challenge experiment, it is ensured that the SQ01Delta meq strain completely loses the pathogenicity and tumorigenicity to the host chicken, and has no immunosuppressive property, and has good biosafety. Animal immune challenge protection evaluation experiment shows that the SQ01Delta meq has good immune protection, and the immune protection index of the parent strain reaches 80.0%. Therefore, the SQ01Delta meq strain constructed by the application can not only be used as a new MD gene deletion vaccine candidate strain, but also be used for the development and utilization of subsequent MD new type gene engineering vaccine.

Description

TECHNICAL FIELD

[0001] The application relates to a MD vaccine candidate strain SQ01Delta meq based on a Marek's disease virus super strong mutant strain and construction and application of meq gene editing deletion, and belongs to the field of animal virology. BACKGROUND

[0002] Marek's disease (MD) is a most important avian immunosuppressive and oncogenic disease caused by early infection with Marek's disease virus (MDV) and results in huge economic losses to the global poultry industry every year. The MD-associated herpesviruses include three different serotypes, serotype I (MDV-1) or Gallid herpesvirus 2 (GaAHV-2), serotype II (MDV-2) or Gallid herpesvirus 3 (GaAHV-3) and serotype III (MDV-3) or Meleagrid herpesvirus 1 (HVT) / (MeAHV-1). Only MDV-1 isolates have pathogenic and oncogenic properties. As the first viral oncogenic disease successfully controlled by vaccine, MD was well controlled in the last decades of the last century. However, the long-term use of MD vaccine and the super strong immune pressure have led to the continuous evolution and increasing virulence of the epidemic MDV strains. In recent years, some very virulent MDV (vv+MDV) strains, especially the very virulent MDV (HV-MDV) mutant strain identified from the cases of MD outbreaks in MD vaccine immunized chicken flocks, have been confirmed to have fully broken through the immune protection of the currently widely used classic MD vaccine, and are likely to be the main cause of the frequent MD outbreaks in the global poultry industry in recent years, causing serious harm to the poultry industry.

[0003] The meq gene is the main oncogenic gene of MDV, with a full length of 1020 bp, and is specific to MDV-1. Studies have shown that the meq gene plays a key role in the occurrence of MDV-induced T cell lymphoma through the expression modification of host and viral genes with other transcription factors. Some scholars found that after knocking out the meq gene of the MD virulent strain by using bacterial artificial chromosome (BAC) and gene homologous recombination technology, the virus no longer has oncogenic effect, which reveals that the meq gene plays a key role in MDV oncogenesis. In subsequent studies, the meq gene was used as the main target for virus genome modification to construct a series of MD gene deletion vaccine candidate strains, such as rMd5Delta meq, Md5BAC Delta Meq Delta LORF9, 686BAC-∆Meq∆vIL8, rMSDelta meq and SC9-1, which have become the focus of MD new vaccine research in the last 20 years of this century.

[0004] In view of the frequent MD cases in vaccine immunized chicken flocks, the current widely used classic MD vaccine has low immunoprotective efficacy against the newly emerging HV-MDV mutant strain, and it is urgent to develop a new more efficient MD vaccine to meet the new challenges of MD prevention and control. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a Marek's disease virus meq gene edited deletion MD vaccine candidate strain SQ01Delta meq and a construction and application thereof based on a super strong variant strain.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0007] The Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Delta meq, the Marek's disease virus meq gene edited deletion vaccine candidate strain is SQ01Delta meq, which has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.45864.

[0008] The parent strain of the vaccine candidate strain SQ01Delta meq is a Marek's disease virus super strong variant strain HNSQ01 isolated from a chicken flock, and the full-length nucleotide sequence of the whole genome of the parent strain is 176043 bp, as shown in SEQ ID NO.1.

[0009] The vaccine candidate strain SQ01Delta meq is a Marek's disease virus super strong variant strain HNSQ01, and two meq alleles in the genome are edited and deleted by a fragment with a length of 925 bp, and the molecular marker sites of gene deletion are 4486-5411 and 133584-134509, respectively.

[0010] The construction method of the Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Delta meq utilizes CRISPR / Cas9 gene editing technology to knockout two meq alleles to construct.

[0011] The Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Delta meq is applied in the preparation of a new type of chicken Marek's disease vaccine.

[0012] The new type of chicken Marek's disease vaccine is a chicken Marek's disease genetic engineering vaccine, a chicken Marek's disease multi-vaccine or a chicken Marek's disease multivalent vaccine constructed by SQ01Delta meq as a vaccine strain, or a chicken Marek's disease genetic engineering vaccine, a chicken Marek's disease multi-vaccine or a chicken Marek's disease multivalent vaccine constructed by SQ01Delta meq as a parent strain.

[0013] The chicken Marek's disease genetic engineering vaccine is a gene deletion vaccine, a gene recombination vaccine or a virus vector vaccine.

[0014] The application includes, but is not limited to, the use of the virus particle, viral nucleic acid DNA or whole genome sequence information of the Marek's disease virus meq gene deletion vaccine candidate strain SQ01Δmeq.

[0015] The present application has the following advantages:

[0016] 1. The meq gene deletion strain SQ01Δmeq of the Marek's disease virus HNSQ01 of the present application is a meq gene edited deletion MD vaccine candidate strain constructed by using the latest isolated HV-MDV variant as the parent strain and using the CRISPR / Cas9 gene editing technology. Through a series of experimental researches such as construction and identification of the meq gene edited deletion strain, 1-day-old SPF chicken pathogenicity analysis and animal immune challenge protection evaluation as a candidate vaccine, it is confirmed that SQ01Δmeq can be used as a new type of high-efficiency MD genetic engineering vaccine candidate strain, which can provide the most comprehensive and reliable experimental basis. The present application develops a new type of gene deletion vaccine strain for the current MDV dominant strain prevalent in chicken flocks, especially the HV-MDV variant, which can provide good immune protection efficiency and fill the gap in the research and development of new generation MD vaccines.

[0017] 2. The parent strain of the Marek's disease virus meq gene deletion vaccine candidate strain SQ01Δmeq provided by the present application is HNSQ01, which is isolated from a layer flock that broke out MD epidemic after vaccination in a poultry farm in 2021, and through a series of MDV pathogenicity analysis and pathogenicity identification experiments such as viral whole genome sequencing, 1-day-old SPF chicken challenge experiment and MD vaccine immune protection challenge experiment, it is defined as a hyper-virulent Marek's disease virus (HV-MDV) variant. This strain can well represent the current MDV dominant strain prevalent in chicken flocks, and the strain source and genetic background are clear.

[0018] 3. The Marek's disease virus meq gene deletion strain SQ01Δmeq of the present application is ensured to completely lose the pathogenicity and tumorigenicity to the host chicken and has no immunosuppressive property and good biosafety through PCR identification, gene cloning sequencing, RT-qPCR analysis, IFA identification and SPF chicken challenge experiment. The immune challenge protection experiment results show that SQ01Δmeq has good immune protection, and the immune protection index (PI) of the parent strain HNSQ01 reaches 80.0%. Therefore, the SQ01Δmeq strain of the present application can not only be used as a new MD gene deletion vaccine candidate strain, but also be used for the development and utilization of subsequent new MD genetic engineering vaccines.

[0019] 4、The virus particle and viral nucleic acid DNA of the Marek's disease virus meq gene deletion vaccine candidate strain SQ01Delta meq can be used as a basis for further construction of more Marek's disease genetic engineering vaccines, such as gene deletion, gene recombination or viral vector vaccines, which can lay an important foundation for the creation of a new generation of MD efficient vaccines, and also can provide a carrier for the genetic engineering recombinant vaccines of other poultry viruses. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Genomic structure of HV-MDV variant strain HNSQ01 and meq gene editing gRNA target point schematic diagram.

[0021] Among them: gRN / gRC, it means that the upstream and downstream gRNA of the meq gene; meq-9F / meq-2R, it means that the upstream and downstream primers of the meq gene; 4486 / 5411 and 133584 / 134509, they are the sites of gRN / gRC cutting two meq alleles of the genome of HV-MDV variant strain HNSQ01.

[0022] Figure 2 PCR amplification identification and sequencing analysis results of SQ01Delta meq gene editing deletion strain.

[0023] Among them: A, it means the PCR amplification identification results of HNSQ01 meq gene editing products; B, it means the PCR amplification identification results of the meq gene editing deletion strain SQ01Delta meq;

[0024] M, it means DNA molecular weight Marker; HNSQ01, it means the parent strain; HNSQ01+gRN / gRC, it means the meq gene editing mixed sample; SQ01Delta meq, it means the meq gene editing deletion strain; CEF, it means the negative control of chicken embryo fibroblast; 50, 100, 150, 200, they mean different infection doses (microliters / well) of HNSQ01; p1, p2, p3, p4, p5, p10, p15, they mean different passage numbers of SQ01Delta meq on CEF.

[0025] Figure 3 Nucleotide sequence analysis atlas of the meq gene PCR amplification product of the SQ01Delta meq gene editing deletion strain.

[0026] Among them: DSB and blue thick arrow, it means the DNA double-strand break of meq gene editing cutting; gRN / gRC, it means the upstream and downstream gRNA targeting the meq gene; dotted arrow, it means the gRNA target point in the meq gene sequence; PAM, it means the protospacer adjacent motif of the target gene.

[0027] Figure 4 Sequencing alignment analysis results of meq gene of SQ01Δmeq gene editing deletion strain and parent strain.

[0028] Wherein: HNSQ01 represents the parent strain; SQ01Δmeq represents the meq gene editing deletion strain; DSB and blue thick arrow represent the DNA double-strand gap of gene editing cleavage; gRN / gRC represents the gRNA targeting the upstream and downstream of meq gene; dotted arrow represents the gRNA target point in the sequence of meq gene; PAM represents the protospacer adjacent motif of target gene.

[0029] Figure 5 IFA identification results of SQ01Δmeq gene editing deletion strain.

[0030] Wherein: HNSQ01 represents the parent strain; SQ01Δmeq represents the meq gene editing deletion strain; pp38 represents the MDV-1 specific pp38 protein monoclonal antibody; Meq represents the MDV-1 specific Meq protein monoclonal antibody; Merge represents the picture coincidence; the picture scale is 50 μm.

[0031] Figure 6 RT-qPCR analysis results of relative expression level of partial virus coding genes of SQ01Δmeq.

[0032] Wherein: HNSQ01 represents the parent strain; SQ01Δmeq represents the meq gene editing deletion strain.

[0033] Figure 7 Statistical analysis results of body weight and immune organ index of SQ01Δmeq challenged chickens.

[0034] Wherein: A represents the average body weight of experimental chickens in each challenge group; B represents the bursa of Fabricius / body weight index of experimental chickens in each challenge group; C represents the thymus / body weight index of experimental chickens in each challenge group; D represents the spleen / body weight index of experimental chickens in each challenge group.

[0035] CEF represents the negative control group; HNSQ01 represents the MDV positive challenge group; SQ01Δmeq represents the meq gene editing deletion strain challenge group. Black asterisk * represents the significant difference (P<0.05) between each group statistically analyzed with the CEF group as the reference; red asterisk * represents the significant difference (P<0.05) between each group statistically analyzed with the HNSQ01 challenge group as the reference.

[0036] Figure 8 Survival curve graph of SQ01Δmeq challenged SPF chickens.

[0037] Wherein: CEF represents the negative cell simulation control group; HNSQ01 represents the MDV infection positive control group; SQ01Δmeq represents the meq gene edited deletion strain infection group. Black asterisk * represents the significant difference (P < 0.05) between the HNSQ01 infection group and the CEF negative simulation control group; red asterisk * represents the significant difference (P < 0.05) between the HNSQ01 infection positive control group and the SQ01Δmeq infection group.

[0038] Figure 9 HE staining diagram of pathological tissue sections of SQ01Δmeq infection chickens.

[0039] Wherein: CEF represents the negative cell simulation control group; HNSQ01 represents the MDV infection positive control group; SQ01Δmeq represents the meq gene edited deletion strain infection group; the picture scale is 20 μm (magnification: 400x).

[0040] Figure 10 Statistical analysis results of body weight and immune organ index of SQ01Δmeq immune infection chickens.

[0041] Wherein: A represents the average body weight of each immune infection group of experimental chickens; B represents the bursa of fabricius / body weight index of each immune infection group of experimental chickens; C represents the thymus / body weight index of each immune infection group of experimental chickens; D represents the spleen / body weight index of each immune infection group of experimental chickens;

[0042] CEF+CEF represents the negative cell simulation control group, that is, after immunizing CEF cells, CEF cells are inoculated to simulate infection; CEF+HNSQ01 represents the infection positive control group, that is, after immunizing CEF cells, HNSQ01 strain is infected; SQ01Δmeq+HNSQ01 represents the immune protection infection group, that is, after immunizing SQ01Δmeq vaccine strain, HNSQ01 strain is infected. Black asterisk * represents the significant difference (P < 0.05) between each group in the statistical analysis with the CEF+CEF negative cell simulation control group as the reference; red asterisk * represents the significant difference (P < 0.05) between each group in the statistical analysis with the CEF+HNSQ01 infection positive control group as the reference.

[0043] Figure 11 Survival curve diagram of SQ01Δmeq immune infection chickens.

[0044] Wherein: CEF+CEF represents the negative cell simulation control group, that is, after immunizing CEF cells, CEF cells are inoculated to simulate an attack; CEF+HNSQ01 represents the attack positive control group, that is, after immunizing CEF cells, the HNSQ01 strain is attacked; SQ01Δmeq+HNSQ01 represents the immunoprotective attack group, that is, after immunizing the SQ01Δmeq vaccine strain, the HNSQ01 strain is attacked. The black asterisk * indicates that the attack positive control group CEF+HNSQ01 and the negative cell simulation control group CEF+CEF are significantly different (P<0.05); the red asterisk * indicates that the attack positive control group CEF+HNSQ01 and the immunoprotective attack group SQ01Δmeq+HNSQ01 are significantly different (P<0.05).

[0045] Figure 12 HE staining diagram of pathological tissue sections of SQ01Δmeq immunized and attacked chickens.

[0046] Wherein: CEF+CEF represents the negative cell simulation control group, that is, after immunizing CEF cells, CEF cells are inoculated to simulate an attack; CEF+HNSQ01 represents the attack positive control group, that is, after immunizing CEF cells, the HNSQ01 strain is attacked; SQ01Δmeq+HNSQ01 represents the immunoprotective attack group, that is, after immunizing the SQ01Δmeq vaccine strain, the HNSQ01 strain is attacked. The black asterisk * indicates that the attack positive control group CEF+HNSQ01 and the negative cell simulation control group CEF+CEF are significantly different (P<0.05); the red asterisk * indicates that the attack positive control group CEF+HNSQ01 and the immunoprotective attack group SQ01Δmeq+HNSQ01 are significantly different (P<0.05). DETAILED DESCRIPTION

[0047] The specific embodiments of the application are further described in detail below with reference to the examples. Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test methods involved are all conventional methods.

[0048] Example 1: Virus isolation and whole genome sequencing and analysis of HNSQ01

[0049] In 2021, a suspected MD case outbreak occurred in a 113-day-old CVI988 / Rispens vaccine immunized layer chicken flock in a farm, and the dead chickens showed hepatosplenomegaly, and some chickens had one or more local tumors or diffuse tumor nodules in internal organs. After conventional PCR and RT-PCR detection excluded chicken infectious anemia, avian leukemia and avian reticuloendotheliosis, the case was finally diagnosed as chicken Marek's disease (MD).

[0050] The sick live chicken was aseptically collected with anticoagulated blood, peripheral blood lymphocytes were extracted, inoculated into CEF monolayer cells prepared in advance, and blind transmission was performed for 1-2 generations. When typical virus plaques appeared, single plaques were picked under a microscope for virus cloning, purification and PCR identification, and finally the MDV isolate HNSQ01 strain was obtained. Through 1-day-old SPF chicken animal challenge experiment and MD vaccine immune challenge protection evaluation experiment, the pathogenic type of the strain was defined as HV-MDV variant strain. The total DNA of the HNSQ01 infected CEF cells was extracted and sent to Shanghai Pisennol Biotechnology Co., Ltd. for viral whole genome sequencing analysis. The final result shows that the full length of the viral genome of the HNSQ01 strain is 176043 bp, and the nucleotide sequence is shown as SEQ ID NO. 1.

[0051] Example two: construction and identification of SQ01Δmeq gene edited deletion vaccine candidate strain

[0052] 1. HNSQ01 virus genome structure and meq gene gRNA design

[0053] According to the HNSQ01 virus genome and its meq gene adjacent sequence, the primer meq-9F / meq-2R for PCR amplification of the meq gene was designed using Primer Premier software; the gRNA sequence gRN / gRC targeting the 5' end and 3' end of the meq gene was designed using Benchling online software, and the meq gene locus and gRNA target point are shown in Figure 1 .

[0054] 2. Editing and analysis of the meq gene of the HNSQ01 strain

[0055] The gRNA oligo sequence targeting the 5' end and 3' end of the MDV-1 meq gene shown in Table 1 (SEQ ID NO. 2-SEQ ID NO. 5) was artificially synthesized, duplexed to form double-stranded, and then cloned into the pX459v2.0 plasmid to construct the gRNA expression plasmid pX459-meq-gRN and pX459-meq-gRC (abbreviated as gRN and gRC, respectively).

[0056] Table 1 gRNA oligo and PCR amplification primer sequence table targeting the meq gene

[0057]

[0058] Note: The underlined part is the Bbs I enzyme digestion site.

[0059] According to the Trans IT-X2 ™The gRNA expression plasmid gRN / gRC was co-transfected into CEF monolayer cells according to the Dynamic Delivery System reagent instruction, and cultured at 38.5°C in a 5% CO2 incubator for 24 h. The p25 generation of the parent strain HNSQ01 passaged in vitro by CEF cells was taken out from liquid nitrogen, and the virus titer was 15257 plaque forming units (PFU) per 100 microliters of virus solution. Different volumes (50 μL, 100 μL, 150 μL, 200 μL) of virus suspension were inoculated. At the same time, CEF cells inoculated with HNSQ01 but not transfected with gRNA plasmid were used as negative controls, and CEF cells not inoculated with HNSQ01 and not transfected with gRNA plasmid were used as blank controls. After 48 h of inoculation, the cells were trypsinized, and 500 μL of medium was used to suspend the cells. Half of the cells were centrifuged to collect the cells, and total DNA was extracted. The meq gene-specific primer pair meq-9F / meq-2R (SEQ ID NO. 6~SEQ ID NO. 7) listed in Table 1 was used for PCR detection to analyze the editing effect of the gRNA plasmid combination on the meq gene of HNSQ01 virus at different inoculation doses.

[0060] The PCR reaction system was as follows: 2x EasyTaq PCR SuperMix 10 μL, 10 μM of upstream and downstream primers 0.5 μL each, 100 ng / μL of sample DNA template 1 μL, and sterilized ultrapure water 8 μL.

[0061] The PCR reaction program was as follows: 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles; 72°C extension for 5 min; 4°C incubation until removal.

[0062] After the reaction, 5 μL of PCR product was taken for analysis and identification by 1% (w / v) agarose gel electrophoresis (150 V, 300 mA, 30 min).

[0063] The electrophoresis results showed that: the CEF blank control group did not amplify any band; the HNSQ01 infected negative control group CEF sample only amplified a specific meq gene wild type band of 1121 bp in size; and the CEF cell samples co-transfected with gRN / gRC plasmid and inoculated with different doses of HNSQ01 all amplified a 196 bp expected gene editing small band in addition to the specific meq gene wild type band of 1121 bp in size. Figure 2 A).

[0064] 3. Purification of SQ01Δmeq gene edited deletion strain and analysis of passage stability

[0065] The CEF cell suspension (about 250 μL) infected with HNSQ01 whose meq gene editing was confirmed by PCR amplification and sequencing was inoculated into the CEF monolayer cells in a 6-well plate according to different doses (5 μL / well, 10 μL / well, 20 μL / well, 40 μL / well), and cultured at 38.5°C in a 5% CO2 incubator for 3-5 days. Then, 72 virus single plaques were randomly selected under a microscope and inoculated into the CEF monolayer cells in a 24-well plate (1 / well). The progeny viruses were cultured for 48 hours, and then the cells were trypsinized and collected by centrifugation. The DNA was extracted from the sample, and the PCR method was used for detection and electrophoresis analysis.

[0066] The cell wells with positive PCR detection were subjected to the second round of virus single plaque cloning and purification. After 2-3 rounds of single cloning and purification, and PCR identification of all virus clones, sequencing analysis was performed again, and the virus was named SQ01Δmeq, which was cultured and stored in liquid nitrogen.

[0067] The SQ01Δmeq was continuously passaged in CEF, and the virus samples of the 1st, 2nd, 3rd, 4th, 5th, 10th and 15th generations were collected. The PCR amplification was used to detect the stability of the meq gene editing deletion. The results showed that the PCR could only amplify the 196 bp mutant meq gene editing product in the DNA of the meq gene editing deletion strain SQ01Δmeq infected cells, and the edited gene sequence was completely consistent with the previous sequencing analysis results. The 196 bp mutant meq gene product could only be amplified in the virus samples of the p1-p15 generations of SQ01Δmeq, indicating that the SQ01Δmeq gene editing deletion strain was stable and no back mutation occurred. Figure 2 B}.

[0068] 4. Identification of SQ01Δmeq gene editing deletion strain

[0069] The 196 bp meq gene editing band was recovered and purified, and then connected to the pMD19-T vector for sequencing ( Figure 3 ), and compared with the meq gene of the HNSQ01 parent strain ( Figure 4 ). The results showed that the gRN / gRC plasmid combination effectively edited the meq gene, and the double-strand break (DSB) occurred precisely at the 3 bases inside the PAM sequence of the 2 gRNAs, and the generated gene mutation was completely consistent with the expectation, i.e., the 925 bp fragments were edited and deleted in the 4486-5411 and 133584-134509 positions of the genome of the parent strain HNSQ01, respectively.

[0070] 5. IFA identification of SQ01Δmeq gene editing deletion strain

[0071] HNSQ01 and SQ01Δmeq strains were inoculated into CEF monolayer cells of 24-well plates, respectively, and cultured in a 38.5℃, 5% CO2 incubator for 3 days, and then fixed with a pre-cooled methanol / acetone (v:v=1:1) solution, 200 μL / well, at room temperature for 10 min; washed with PBST for 3 times, added with 5% (w / v) skim milk-containing PBST blocking, 200 μL / well, at 37℃ for 30 min; washed with PBST for 3 times, added with 1:5000 diluted MDV-Meq monoclonal antibody (self-made) for incubation, 100 μL / well, at 37℃ for 30 min; washed with PBST for 3 times, added with Dylight 594 goat anti-mouse IgG (1:1000) for incubation, 100 μL / well, at 37℃ for 30 min; washed with PBST for 3 times, added with 1:1000 diluted Dylight 488-labeled MDV-pp38 monoclonal antibody (self-made) for incubation, 100 μL / well, at 37℃ for 30 min; finally washed with PBST for 3 times, and observed under a fluorescence microscope.

[0072] The results show that: the CEF cells infected by HNSQ01 and SQ01Δmeq can form MDV virus plaques with similar morphology, and can specifically bind with the green fluorescent labeled MDV-pp38 monoclonal antibody, and the red fluorescent indicated MDV-Meq monoclonal antibody only binds with the virus plaques in the HNSQ01 infected cells, and no specific red fluorescent staining virus plaques are observed in the SQ01Δmeq infected cells Figure 5 ), indicating that the meq gene of SQ01Δmeq is completely edited and deleted, and no longer expresses Meq protein.

[0073] 6. RT-qPCR identification of SQ01Δmeq gene edited deletion strain

[0074] HNSQ01 and SQ01Δmeq were inoculated into 6-well plate CEF monolayer cells, respectively, and cultured in a 38.5℃, 5% CO2 incubator for 3-5 days, and then observed under a microscope to observe the virus plaques, and then the cells were trypsinized and centrifuged to collect the cells, and total RNA was extracted by TRIzol, and cDNA was prepared by TaKaRa TM 5×PrimeScript RT Master Kit. The prepared cDNA was used as a template for qPCR amplification by Fast Universal SYBR ® Green Master Kit, and the relative expression levels of 6 MDV genes ICP4, meq, gB, pp38, RLORF4 and RLORF5a were determined. The primers for the 6 MDV genes are shown in Table 2 (SEQ ID NO. 8-SEQ ID NO. 19).

[0075] Table 2. MDV gene RT-qPCR primer sequences

[0076]

[0077] The results show that, using ICP4 as the internal reference, it is found that the above-mentioned genes encoded by the parent strain HNSQ01 can be normally expressed in the CEF cells infected by the virus; the gB, pp38, RLORF4 and RLORF5 encoded by SQ01Δmeq are also detected to be normally expressed, but the expression of meq gene is not detected Figure 6 , further confirming that the meq gene in the viral genome of SQ01Δmeq is completely edited and deleted.

[0078] Example Three: Pathogenicity analysis of SQ01Δmeq gene deletion vaccine candidate strain

[0079] 1. Viral titer determination of HNSQ01 and SQ01Δmeq

[0080] The HNSQ01 and SQ01Δmeq virus seeds stored in liquid nitrogen were quickly dissolved in a 37°C water bath, and then centrifuged at 1000 r / min for 5 min. The cell freezing liquid was discarded, and the cells were resuspended in 1 mL of M199 medium containing 1% (v / v) FBS (fetal bovine serum) and 10% (v / v) TPB (tryptone phosphate broth), respectively. After mixing thoroughly, 900 μL was aspirated, and two virus seeds were diluted by 2 -1 ~2 -12 fold in M199 medium containing 1% (v / v) FBS and 10% (v / v) TPB, respectively; then 2 -6 ~2 -12 fold dilution of the virus liquid was transferred to the CEF monolayer cells in a 48-well plate, 300 μL / well, 3 repeats for each dilution, and the last column of cell wells was directly supplemented with 1% (v / v) FBS and 10% (v / v) TPB M199 medium as a negative control.

[0081] The cells were placed in a 38.5℃, 5% CO2 incubator for 3-4 days, and then the typical virus plaques were observed under a microscope. Then, 3 dilution wells with moderate number of plaques (15-30 plaques per well) were selected, and the virus plaques were stained with MDV-pp38 monoclonal antibody and DyLight 488 goat anti-mouse IgG (1:1000) secondary antibody according to the IFA method described in Example 2. Finally, the virus plaques were counted under an inverted fluorescence microscope. The average number of virus plaques in the 3 repeated wells of each dilution was multiplied by the corresponding dilution factor to calculate the virus titers of HNSQ01 and SQ01Δmeq, which were 1.53 x 10 4 PFU / 100 μL and 4.51 x 10 4 PFU / 100 μL, respectively.

[0082] 2. Animal inoculation experiment of SQ01Δmeq

[0083] 90 one-day-old white leihang SPF chickens were randomly divided into 3 groups, which were used as HNSQ01 challenge group, SQ01Δmeq challenge group and CEF negative control group, respectively. The challenge groups were inoculated intraperitoneally at a dose of 2000 PFU / 200 μL per chicken, and the negative control group was injected with the same amount of CEF negative cells. The chickens were raised in a positive pressure chicken isolator. The clinical symptoms, morbidity and mortality of the chickens in each group were observed daily after challenge, and the tumor occurrence was observed by necropsy. The survival curve was drawn. Every week, 5 chickens were randomly selected from each group for weighing (3 males and 2 females). On the 14th day and the 21st day after challenge, 4 chickens from each group were weighed and then euthanized, and the thymus, bursa of Fabricius and spleen immune organs were collected, weighed and recorded to calculate the immune organ index: [immune organ weight (g) / body weight (g)] x 100. At the end of the experiment on the 77th day, all the surviving chickens were euthanized and necropsied to observe the tumor occurrence in internal organs. Meanwhile, 3 chickens were randomly selected from each group, and the liver and spleen were collected to prepare tissue sections, which were stained with HE to observe the histopathological changes.

[0084] 3. Results of animal pathogenicity analysis of SQ01Δmeq

[0085] The results of animal experiment showed that the statistical analysis results of the body weight changes of the chickens in each group with days post inoculation (dpi) were as follows: Figure 7As shown in Fig. 2A, there was a significant difference between the HNSQ01 group and the CEF negative control group (P < 0.05) and between the HNSQ01 group and the SQ01Δmeq group from 14 dpi to 63 dpi. Except for 70 dpi, there was no significant difference between the SQ01Δmeq group and the negative control group (P > 0.05), indicating that SQ01Δmeq did not affect the weight gain of the experimental chickens. Throughout the experimental period, the weight of the HNSQ01 positive challenge group was significantly different from the other two groups (P < 0.05).

[0086] The statistical analysis results of the immune organ indices of the chickens in each group are shown in Figs. 3A, 3B, 3C and 3D. Figure 7 As shown in Figs. 3B, 3C and 3D, at 14 dpi and 21 dpi, the thymus index, bursa index and spleen index of the HNSQ01 group were significantly different from those of the CEF negative group (P < 0.05), and the SQ01Δmeq group was not significantly different from the CEF negative group (P > 0.05) except for the spleen index at 14 dpi. The bursa index at 14 dpi and 21 dpi and the thymus index at 14 dpi of the HNSQ01 group were significantly different from those of the SQ01Δmeq group (P < 0.05), and there was no significant difference between the other groups (P > 0.05). The above data show that SQ01Δmeq has little effect on the central immune organs (thymus and bursa) of infected chickens and does not cause obvious immune suppression.

[0087] During the entire animal challenge experiment period, the early deaths of the experimental chickens in the first week were probably due to abdominal infection or other stress caused by virus inoculation, so these death numbers were all excluded from the statistics. In addition, the remaining chickens were counted according to the number of deaths of each challenge group recorded daily, and the survival curves of each group were plotted, as shown in Fig. 4. Figure 8 As shown in Fig. 4, during the entire experimental period, until the end of the experiment at 77 d, no deaths were observed in the CEF negative control group and the SQ01Δmeq challenge group, while all the experimental chickens in the HNSQ01 positive control challenge group had died by 67 d. The survival curves showed that there were significant differences between the CEF negative control group and the SQ01Δmeq challenge group and between the HNSQ01 positive control challenge group (P < 0.05).

[0088] At the end of the experiment, in addition to observing the occurrence of tumors in the experimental chickens, 3 chickens were randomly selected from each group to collect liver and spleen, prepare tissue sections and perform HE staining, and the pathological observation results are shown in Figs. 5A, 5B and 5C. Figure 9As shown: the liver and spleen tissues of 3 chickens in the HNSQ01 challenge group were seen large area of typical lymphocyte infiltration, liver cells and spleen cells lost the original cell morphology, cell nucleus swelling and showed multipolar; while the liver and spleen of 6 experimental chickens in the SQ01Δmeq challenge group and the CEF negative control group were not seen lymphocyte infiltration and other pathological phenomena.

[0089] In summary, the meq gene completely edited deletion MDV vaccine candidate strain SQ01Δmeq is successfully constructed by taking HNSQ01 as the parent strain, the animal safety evaluation results show that the gene deletion strain completely loses the pathogenicity and tumorigenicity to the host chicken, and no typical immune organ atrophy is seen after inoculating SPF chickens, and can be used as a MD candidate vaccine strain. The gene deletion strain SQ01Δmeq has been preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC NO.45864, and the preservation time is January 17, 2024.

[0090] Example 1, immune challenge protection evaluation test of SQ01Δmeq gene deletion vaccine candidate strain

[0091] 1, SQ01Δmeq immune challenge protection test scheme

[0092] 81 1-day-old white leihang SPF chickens were randomly divided into 3 groups, the first group was CEF+CEF negative cell simulation control group, the second group was CEF+HNSQ01 challenge positive control group, and the third group was SQ01Δmeq+HNSQ01 immune protection challenge group. At 1 day old, all experimental chickens were injected subcutaneously on the nape, the third group was inoculated with 3000 PFU / 200 μL per chicken, and the first and second groups of experimental chickens were inoculated with equal amount of CEF cells, respectively, and were raised in a positive pressure isolator. After 7 days of vaccine immunization, the second and third groups of experimental chickens were inoculated with HNSQ01 (1000 PFU / 200 μL per chicken) intraperitoneally, and the first group of negative control group was inoculated with equal amount of CEF cells.

[0093] The above each group of experimental chickens were separately fed in positive pressure isolator, observed and recorded the incidence and death of each group of experimental chickens daily, and drew the survival curve of each group. Every week, 5 chickens (3 males and 2 females) were randomly selected from each group to weigh and record the growth. On the 14th day and the 21st day after the challenge, 5 chickens were randomly selected from each group to be euthanized, and the immune organs such as thymus, bursa of fabricius and spleen were collected, weighed and recorded to calculate the immune organ index, and the immune organ index = [immune organ weight (g) / body weight (g)] x 100. At the same time, the dead chickens were dissected to observe the occurrence of internal tumors. Until the end of the experiment on the 70th day after the challenge, all the experimental chickens that were still alive were euthanized, and the occurrence of internal tumors was observed and recorded. At the same time, 3 chickens were randomly selected from each group to collect the spleen, prepare tissue sections and perform HE staining to observe the histopathological changes.

[0094] 2. Body weight and immune organ index of SQ01Δmeq immunized and challenged experimental chickens

[0095] The statistical analysis of the body weight and immune organ index of each group of experimental chickens at different time points after the challenge showed that the statistical analysis results of the body weight of each group of chickens were as shown in Table 2. Figure 10 A, the body weight of the CEF+HNSQ01 challenge positive control group and the CEF+CEF negative cell simulation control group at 7 dpi~70 dpi had significant differences (P<0.05) except for the 3 time points of 21 dpi, 28 dpi and 63 dpi; the body weight of the CEF+HNSQ01 challenge positive control group and the SQ01Δmeq+HNSQ01 immunoprotection challenge group had significant differences (P<0.05) at 7 dpi, 14 dpi, 35 dpi, 42 dpi, 49 dpi and 56 dpi; and the body weight of the SQ01Δmeq+HNSQ01 immunoprotection challenge group and the CEF+CEF negative cell simulation control group had no significant difference (P>0.05) at any time point during the whole experimental period. From the analysis of the body weight data during the whole experimental period, the influence of the SQ01Δmeq+HNSQ01 immunoprotection challenge group on the body weight increase of the experimental chickens was significantly less than that of the CEF+HNSQ01 challenge positive control group, and there was no significant difference between the SQ01Δmeq+HNSQ01 immunoprotection challenge group and the CEF+CEF negative cell simulation control group, which indicated that the SQ01Δmeq immunization significantly reduced the inhibition of the HNSQ01 challenge on the body weight increase of the experimental chickens.

[0096] The statistical analysis results of the immune organ index of each group of experimental chickens were as shown in Table 3. Figure 10As shown in (B, C, and D): At 14 dpi and 21 dpi, the bursa of Fabricius index, thymus index, and spleen index were significantly different between the CEF+HNSQ01 challenge-positive control group and the CEF+CEF negative cell simulated control group (P<0.05); between the SQ01Δmeq+HNSQ01 immune protection challenge group and the CEF+CEF negative cell simulated control group, there were no significant differences in the bursa of Fabricius index and thymus index (P>0.05), but a significant difference in the spleen index (P<0.05); between the CEF+HNSQ01 challenge-positive control group and the SQ01Δmeq+HNSQ01 immune protection challenge group, the bursa of Fabricius index and thymus index were significantly different at 14 dpi and 21 dpi (P<0.05), but the spleen index was not significantly different (P>0.05). These data indicate that after immunization with SQ01Δmeq, challenge with HNSQ01 did not cause atrophy of the bursa of Fabricius and thymus, the central immune organs of the infected chickens.

[0097] 3. Analysis of tumor occurrence in chickens subjected to SQ01Δmeq immune challenge experiment

[0098] Throughout the entire animal immunization and challenge experiment, early deaths in the first week of each group of experimental chickens were likely due to intra-abdominal infection or other stressors, and therefore these deaths were excluded from the statistics. In addition, throughout the subsequent experimental period, the mortality of each group of experimental chickens was observed and recorded daily. Chickens that died from illness were promptly necropsed, and the occurrence of gross tumors was observed and recorded until the end of the 70-day challenge experiment. Surviving chickens in each group were then euthanized and necropsed to observe and record the occurrence of gross tumors in internal organs. The final statistical analysis results are shown in Table 3: By the end of the animal experiment 70 days after challenge, the CEF+CEF negative cell simulated control group showed no illness or death, and no grossly observable internal organ tumors were found in any of the surviving chickens; the tumor incidence rate in the CEF+HNSQ01 challenge positive control group was 76.5%; the tumor incidence rate in the SQ01Δmeq+HNSQ01 immune protection challenge group was 13.3%, significantly lower than that in the CEF+HNSQ01 challenge positive control group (P<0.05).

[0099] Table 3. Statistical table of MD tumor incidence in chickens challenged after SQ01Δmeq immunization.

[0100]

[0101] 4. Analysis results of morbidity, mortality and immune protection index of SQ01Δmeq immunized challenge chickens

[0102] The MD morbidity, mortality and immune protection index (PI) of each group of experimental chickens were compared, and the results are shown in Table 4: until the end of the animal experiment 70 days after the challenge, the MD morbidity and mortality of the CEF+CEF negative cell simulation control group were both 0%; the MD morbidity and mortality of the CEF+HNSQ01 challenge positive control group were 100% and 88.2%, respectively; the MD morbidity and mortality of the SQ01Δmeq+HNSQ01 immune protection challenge group were 20.0% and 13.3%, respectively, both of which were significantly lower than those of the CEF+HNSQ01 challenge positive control group (P<0.05). The immune protection index (PI) of SQ01Δmeq immune protection against HNSQ01 challenge was finally calculated to be 80.0%.

[0103] Table 4 MD morbidity, mortality and immune protection index of chickens after SQ01Δmeq immunization challenge

[0104]

[0105] 5. Survival curve analysis results of SQ01Δmeq immunized and challenged experimental chickens

[0106] During the entire animal immunization and challenge experiment period, the number of early deaths of the first week of each group of experimental chickens was deducted. According to the number of deaths of each immune challenge group recorded daily, the survival curves of each group were drawn, as shown in Figure 11 : Until the end of the experiment 70 days after the challenge, no deaths were observed in the CEF+CEF negative cell simulation control group, and only 2 chickens survived in the CEF+HNSQ01 challenge positive control group. The SQ01Δmeq+HNSQ01 immune protection challenge group had 2 deaths throughout the experimental period. The survival curves showed that the CEF+CEF negative cell simulation control group and the SQ01Δmeq+HNSQ01 immune protection challenge group were significantly different from the CEF+HNSQ01 challenge positive control group (P<0.05).

[0107] 6. Results of histopathological changes of SQ01Δmeq immunized and challenged experimental chickens

[0108] Until the end of the experiment, in addition to observing the tumor occurrence of the experimental chickens, 3 chickens from each group were randomly selected to collect spleen tissue for histological sectioning and HE staining, and the results are shown in Figure 12 : The spleen tissue structure of the 3 chickens in the CEF+CEF negative cell simulation control group was normal; the spleen tissue of the 3 chickens in the CEF+HNSQ01 challenge positive control group showed different degrees of lymphocyte infiltration, and the splenocytes lost their original cell morphology, with swollen nuclei and multipolarization; while the spleen of the 3 chickens in the SQ01Δmeq+HNSQ01 immune protection challenge group showed no obvious lymphocyte infiltration and other pathological phenomena.

[0109] In summary, based on the HV-MDV variant HNSQ01 and CRISPR / Cas9 gene editing technology, the application successfully constructs a vaccine candidate strain SQ01Δmeq of Marek's disease virus meq gene editing deletion. The strain not only completely loses the pathogenicity and tumorigenicity to the host chicken and has good biosafety, but also can be used as a MD candidate vaccine strain. Animal immune challenge protection evaluation experiment shows good immune protection effect. The immune protection index (PI) of the strain to the current popular HV-MDV variant HNSQ01 challenge reaches 80.0%, which exceeds the standard of PI index reaching more than 75% required by MD vaccine new veterinary drug declaration and registration, and has good market application prospect. Therefore, the SQ01Δmeq strain constructed by the application can be used as a new MDV meq gene deletion vaccine candidate strain, and is used for subsequent development and utilization of new type high-efficiency MD genetic engineering vaccine.

[0110] Using SQ01Δmeq as the parent strain, a series of MDV double gene deletion, multi-gene deletion MDV vaccine strains, and even genetic engineering recombinant vaccines expressing other poultry disease antigen genes can be constructed by using the above technical route and method to create a new generation of high-efficiency MD vaccine, which will not be listed one by one in the application.

Claims

1. A Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Δmeq, characterized in that, The Marek's disease virus meq gene edited deletion vaccine candidate strain is SQ01Δmeq, which has been preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC NO. 45864.

2. Use of the Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Δmeq of claim 1 in the preparation of a vaccine for chicken Marek's disease.

3. Use according to claim 2, wherein the compound is ###0002### The chicken Marek's disease vaccine is a genetically engineered vaccine for chicken Marek's disease, a multi-link vaccine for chicken Marek's disease or a multivalent vaccine for chicken Marek's disease constructed by using SQ01Δmeq as a vaccine strain; or a genetically engineered vaccine for chicken Marek's disease, a multi-link vaccine for chicken Marek's disease or a multivalent vaccine for chicken Marek's disease constructed by using SQ01Δmeq as a parent strain.

4. The use according to claim 3, wherein the compound is ###0002### The genetically engineered vaccine for chicken Marek's disease is a gene deletion vaccine, a gene recombination vaccine or a viral vector vaccine.

5. The use according to claim 4, wherein the compound is ###0002### The use is selected from the use of virus particles and viral nucleic acid DNA of the Marek's disease virus meq gene deletion vaccine candidate strain SQ01Δmeq.

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