Recombinant Marek's virus vector vaccine strain for expressing chicken infectious laryngotracheitis virus gD protein and application of recombinant Marek's virus vector vaccine strain

Abstract

The invention provides a recombinant Marek's virus vector vaccine strain for expressing chicken infectious laryngotracheitis virus gD protein and application of the recombinant Marek's virus vector vaccine strain, and belongs to the technical field of veterinary biological products. According to the recombinant Marek virus vector vaccine strain, a TK promoter is used as a starting element of gD protein of infectious laryngotracheitis virus, HSV TK polyA is used as a terminator, a gD gene expression box is constructed, and the gD gene expression box is inserted into a UL3 / 4 or UL45 / 46 site of an I-type MDV SC9-1 genome serving as a vector to construct the recombinant Marek virus vector vaccine strain. The vaccine strain can simultaneously induce an organism to generate cellular immune response and humoral immune response, so that the immunodeficiency of an inactivated vaccine and a subunit vaccine is avoided, and meanwhile, the side reaction and virulence return of a live vaccine are overcome; the bivalent vaccine of MDV and ILTV can be formed at the same time, the dosage of the vaccine is small, the immune protection duration is long, the effect is good, an adjuvant does not need to be added, and the cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a recombinant Marek's virus vector vaccine strain expressing infectious laryngotracheitis virus gD protein and a use thereof. Background Art

[0002] Infectious laryngotracheitis (ILT) is an acute respiratory infectious disease of chickens caused by infectious laryngotracheitis virus (ILTV), which causes great economic losses to the global poultry industry.

[0003] ILT is characterized by swelling and bleeding of the laryngeal and tracheal mucosa, which can cause erosion in severe cases. It was called "tracheo-laryngitis" and "avian diphtheria" at the time. The disease can show different clinical symptoms depending on the virulence of the virus strain that causes the disease. Mild clinical symptoms mainly include tearing, runny nose, inflammation of the infraorbital sinus and conjunctiva of the affected birds, while severe cases often show dyspnea, rales, bleeding of the tracheal epithelial mucosa and fibrinous exudate. After chickens immunized with attenuated vaccines and sick chickens recover, ILTV can establish lifelong latent infection in the central nervous system of infected chickens, especially in the trigeminal ganglion. At the same time, the virulence of ILTV will be significantly enhanced after continuous passage among chickens.

[0004] The prevention and control of ILT mainly relies on vaccination. Chicken embryo-derived and tissue culture-derived vaccines produced in the 1960s have been widely used to control the global ILT epidemic. However, as the attenuated live vaccine strains continue to spread in chickens, the virulence has increased. It has been reported that the ILTV chicken embryo-derived vaccine strain can undergo genetic mutations and increase virulence through continuous passage between chickens. In order to overcome the problems associated with attenuated live vaccines, scientists have used genetic engineering technology to develop turkey herpesvirus (HVT) and fowlpox virus (FPV) vector vaccines that can express the main immunogenic proteins of ILTV. HVT-ND-ILT and HVT-ILT promoted by Intervet are widely used abroad, but not on the market in China. According to literature reports, HVT-ND-ILT, HVT-ILT and other HVT vector vaccines have limited immune protection effects and cannot achieve 100% immune protection against ILT. In addition, the immune protection effect of HVT immunization against the strong toxin of Marek's virus is not ideal. The most widely used ones in China are the FPV-ILT developed by Harbin Veterinary Research Institute and the FPV-ILT developed and produced by Shihua Company. However, due to interference from FPV maternal antibodies, etc., its immune protection effect is limited when used alone for clinical immunization.

[0005] Marek's disease (MD) is a lymphoproliferative tumor disease of chickens caused by Marek's disease virus (MDV), and is also one of the major infectious diseases of poultry prevalent worldwide. MDV is a cell-associated virus, divided into three serotypes: type I is tumorigenic MDV, type II is non-tumorigenic MDV, and type III is antigenically related to herpesvirus of turkeys (HVT). HVT vaccine has been widely used to prevent MDV virulent (vMDV) infection because it can be freeze-dried and stored and is easy to use. However, since the late 1970s, there have been very virulent MDV (vvMDV) and very virulent (vv+MDV) strains that HVT vaccine cannot effectively protect against. The protective effect of serotype I MDV vaccine strains against vMDV, vvMDV and vv+MDV strains is significantly better than that of HVT vaccine. As the virulence of MDV continues to increase, the traditional attenuated vaccine CVI988 / Rispense strain is often unable to provide complete protection against vv+MDV, and new genetically engineered vaccines have become the trend for preventing vv+MDV.

[0006] The parental virus GX0101 of the genetically engineered vaccine SC9-1 was isolated in Guangxi Zhuang Autonomous Region, China in 2001. Later, Cui Zhizhong and others from Shandong Agricultural University knocked out the pathogenic meq gene to obtain the attenuated SC9-1 vaccine strain. SC9-1 has obtained the Agricultural Genetically Modified Organism Safety Approval Certificate (Nongjianzhengzi (2014) No. 23) and the New Veterinary Drug Registration Certificate ((2019) New Veterinary Drug Certificate No. 25). The advantages of using the SC9-1 strain as a vector to construct a genetically engineered vaccine are: (1) SC9-1 is a herpes virus that can cause persistent infection after infecting chickens. Therefore, after one immunization, the exogenous antigen can be continuously expressed, stimulating the body to produce a strong immune response; (2) SC9-1 has strict cell binding properties, and the way it spreads in the body is through direct contact between cells, which is not interfered by maternal antibodies; (3) The SC9-1 genome is 180kb long, and several sites have been confirmed to be non-essential regions for viral replication, which is conducive to the simultaneous insertion of multiple exogenous Genetically constructed multivalent vaccine or multi-component vaccine; (4) SC9-1 has obtained the new veterinary drug registration certificate number and has been widely promoted and applied in the market, with guaranteed safety; (5) SC9-1 as an MDV vector vaccine can be administered in ovo or at 1 day old to induce early immune protection, and express exogenous antigens to stimulate the animal body, which can produce both humoral immunity and cellular immunity, with a relatively ideal immune protection effect; (6) Compared with type I CVI988 strain and type III HVT strain, SC9-1 strain has a broader application prospect because it can provide better immune protection against Marek's disease.

[0007] In view of the problems such as the serious side effects and insufficient efficacy of ILTV attenuated live vaccine and the commercialized ILTV-related live vector vaccine, it is urgent to develop a new vaccine to prevent and control chicken infectious laryngotracheitis and chicken Marek's disease. The SC9-1 strain has good safety and strong protection against MDV, so it is used as the preferred research object of the present invention. Although there are relevant research reports on SC9-1 as a carrier to express chicken infectious bursal disease virus VP2 protein and avian influenza virus HA protein, there is no research on the live vector vaccine of SC9-1 expressing chicken infectious laryngotracheitis related protein. And as known to those skilled in the art, using the same live vector to express different foreign proteins, in order to achieve the best effect, the insertion site of the foreign gene, the selection of the promoter and the expression cassette, the optimization method of the gene, etc. also need a long exploration. Summary of the invention

[0008] The present invention provides a recombinant Marek's virus vector vaccine strain expressing the gD protein of infectious laryngotracheitis virus of chickens and its use. The vaccine strain can simultaneously induce the body to produce cellular immunity and humoral immune response, avoid the immunodeficiency of inactivated vaccines and subunit vaccines, and can simultaneously constitute a two-in-one vaccine of MDV and ILTV, with a small amount of vaccine, a long duration of immune protection, good effect, no need to add adjuvant, and reduced cost.

[0009] In order to achieve the above-mentioned object, the present invention provides a recombinant Marek's virus vector vaccine strain expressing the gD protein of infectious laryngotracheitis virus of chickens, wherein a TK promoter is used as the promoter element of the gD protein of infectious laryngotracheitis virus, and HSVTK polyA is used as the terminator to construct a gD gene expression cassette, and the gD gene expression cassette is inserted into the UL3 / 4 or UL45 / 46 site of the type I MDV SC9-1 genome as a vector to construct a recombinant Marek's virus vector vaccine strain expressing the gD protein of infectious laryngotracheitis virus.

[0010] Preferably, the nucleotide sequence of the TK promoter is shown in SEQ ID NO: 1, the nucleotide sequence of the HSV TK polyA is shown in SEQ ID NO: 2, and the nucleotide sequence of the gD gene is shown in SEQ ID NO: 3.

[0011] Preferably, the amino acid sequence of the gD protein is shown in SEQ ID NO:4.

[0012] Preferably, the recombinant Marek's virus vector vaccine strain is a live attenuated vector vaccine.

[0013] The present invention provides a use of the recombinant Marek's virus vector vaccine strain according to any of the above technical solutions in the preparation of a medicament for simultaneously attacking and protecting against infectious laryngotracheitis virus and Marek's disease virus.

[0014] The present invention provides a use of the recombinant Marek's virus vector vaccine strain according to any one of the above technical solutions in the preparation of a medicament for preventing animals from being simultaneously infected by infectious laryngotracheitis virus and Marek's disease virus.

[0015] Preferably, the recombinant Marek's virus vector vaccine strain is administered by subcutaneous injection in the neck, with an immunization dose of 3000 PFU / 0.2 ml.

[0016] Preferably, 28 days after immunization, the highly virulent Wanggang strain of infectious laryngotracheitis virus is challenged by intratracheal injection, with 10 4.0 EID 50 / 0.2ml, the protection rate reaches 100%.

[0017] Preferably, 6 days after immunization, the chicken is challenged with the super virulent strain of Marek's virus Md5 by intraperitoneal injection, with 1000 PFU / 0.2 ml per chicken, and the protection rate reaches 100%.

[0018] The present invention provides a DIVA vaccine, which is prepared by using the recombinant Marek's virus vector vaccine strain described in any one of the above technical solutions.

[0019] Compared with the prior art, the advantages and positive effects of the present invention are:

[0020] 1. The present invention successfully screened out the TK promoter and its gD protein expression cassette, screened out the UL3 / 4 and UL45 / 46 sites that can stably and efficiently express foreign proteins, and developed a live vector vaccine strain that can provide 100% protection against both ILTV and MDV and is absolutely safe for chicks. The vector vaccine strain uses the SC9-1 attenuated strain as a vector to express the gD protein of ILTV, which can perfectly avoid the safety risks brought by the above-mentioned ILTV live vaccine.

[0021] 2. The vector vaccine constructed by the present invention contains only the gD protein of ILTV. When used in conjunction with an ILTV gB protein antibody detection kit, it can distinguish between wild virus infection or attenuated live vaccine immunization and live vector vaccine immunization, which is beneficial for monitoring the infection of ILTV at any time and achieving population purification of ILTV.

[0022] 3. Compared with conventional vaccines, the recombinant virus live vector vaccine screened by the present invention also has the following advantages: (1) it can simultaneously activate the body's cellular immunity and humoral immunity, avoiding the immune deficiency of inactivated vaccines and subunit vaccines, while overcoming the side effects and virulence reversion of ILTV live vaccines; (2) it can simultaneously constitute a bivalent vaccine: it can reduce production costs, simplify immunization procedures, and overcome the interference between different virus attenuated vaccines, and can simultaneously prevent and control infectious laryngotracheitis and Marek's disease in chickens; (3) the vaccine dosage is small, the immune protection lasts for a long time and the effect is good, no adjuvant needs to be added, and the breeding cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 PCR identification of the transfer vector plasmid provided in the embodiment of the present invention, wherein M: DL5000DNA Marker; 1: PCR identification of pBlu-TK promoter-gD-PolyA-Kana; 2: PCR identification of pBlu-gD / gI promoter-gD / gI-PolyA-Kana;

[0024] Figure 2 PCR identification of the infectious clone plasmid provided in the embodiment of the present invention, wherein M: DL5000DNAMarker; 1-3: PCR identification of SC9-TK-Kana (UL3 / 4), SC9-TK-Kana (UL41) and SC9-TK-Kana (UL45 / 46); 4-6: PCR identification of SC9-TK (UL3 / 4), SC9-TK (UL41) and SC9-TK (UL45 / 46); 7-9: PCR identification of SC9-gD-Kana (UL3 / 4), SC9-gD-Kana (UL41) and SC9-gD-Kana (UL45 / 46); 10-12: PCR identification of SC9-gD (UL3 / 4), SC9-gD (UL41) and SC9-gD (UL45 / 46);

[0025] Figure 3 Genetic stability identification of the recombinant virus provided in the embodiment of the present invention (TK promoter), wherein M: DL5000 DNA Marker; 1-5: PCR identification of the 1st, 5th, 10th, 15th and 20th generations of the recombinant virus SC9-gD (TK, UL3 / 4); 6-10: PCR identification of the 1st, 5th, 10th, 15th and 20th generations of the recombinant virus SC9-gD (TK, UL41); 11-15: PCR identification of the 1st, 5th, 10th, 15th and 20th generations of the recombinant virus SC9-gD (TK, UL45 / 46);

[0026] Figure 4Genetic stability identification of the recombinant virus provided in the embodiment of the present invention (gD / gI promoter), wherein M: DL5000 DNA Marker; 1-5: PCR identification of the 1st, 5th, 10th, 15th and 20th generations of the recombinant virus SC9-gD (gD / gI, UL3 / 4); 6-10: PCR identification of the 1st, 5th, 10th, 15th and 20th generations of the recombinant virus SC9-gD (gD / gI, UL41); 11-15: PCR identification of the 1st, 5th, 10th, 15th and 20th generations of the recombinant virus SC9-gD (gD / gI, UL45 / 46);

[0027] Figure 5 The proliferation curve of the recombinant virus provided in the embodiment of the present invention in CEF cells;

[0028] Figure 6 The IFA provided in the embodiment of the present invention identifies the expression of exogenous proteins. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1 Selection of foreign gene insertion sites

[0031] The present invention firstly screens the exogenous gene insertion site of the MDV vector vaccine parent strain SC9-1.

[0032] The details are as follows:

[0033] The EGFP fluorescent gene expression cassette CMV-EGFP-BGHPolyA was constructed with CMV as the promoter and BGH PolyA as the terminator. Different positions in the SC9-1 genome were selected as the exogenous gene insertion sites. After adding 50bp homology arm sequences at both ends of the EGFP expression cassette, they were electroporated to the US2 site, US7 / 8 site, US10 site, UL3 / 4 site, UL7 / 8 site, UL21 / 22 site, UL22 / 23 site, UL41 site, UL45 / 46 site and UL55 / LORF 10 site of SC9-1, infectious clones were constructed and recombinant viruses were rescued by transfection, and they were continuously passaged in CEF cells to the 15th generation.

[0034] The results are shown in Table 1. Inserting the EGFP gene expression cassette at the above-mentioned 10 sites can rescue the recombinant virus and express fluorescent protein. Among them, after the insertion of foreign genes at sites US2 and UL7 / 8, the replication of the recombinant virus in CEF cells was seriously affected, and the replication ability was significantly reduced compared with the parent strain SC9-1; after the insertion of foreign genes at sites US10 and UL21 / 22, the fluorescence intensity of expression was weak, indicating that the ability of these two sites to express foreign proteins was weak; after the insertion of foreign genes at sites US7 / 8, UL22 / 23 and UL55 / LORF 10, the viral replication ability was slightly affected, but the recombinant virus at site US7 / 8 was passaged to the third generation, the recombinant virus at site UL22 / 23 was passaged to the eighth generation, and the recombinant virus at site UL55 / LORF was passaged to the eighth generation. 10 The inserted foreign gene was found to be missing after the recombinant virus was passed down to the fifth generation, indicating poor genetic stability; after the foreign gene was inserted into the sites UL3 / 4, UL41 and UL45 / 46, the replication ability of the recombinant virus was similar to that of the parent strain SC9-1, and no gene deletion, insertion or recombination was found after the 15th generation, and the fluorescent gene could be continuously and efficiently expressed, indicating good genetic stability. Therefore, in the subsequent invention research, the three positions of UL3 / 4, UL41 and UL45 / 46 were selected as the insertion sites of the target protein.

[0035] Table 1 Screening results of different exogenous gene insertion sites

[0036]

[0037]

[0038] Example 2 Screening of different promoters for gD (ILTV) protein expression cassette

[0039] The present invention further attempts to select promoters such as CMV, mCMV, EF1α, SV40 and CAG (composite promoter) that are well known to researchers in the field and have been published and commonly used as expression elements of gD protein, and construct expression cassettes CMV-gD-PolyA, mCMV-gD-PolyA, EF1α-gD-PolyA, SV40-gD-PolyA and CAG-gD-PolyA, respectively.

[0040] Select the 3 gene insertion sites UL3 / 4, UL41 and UL45 / 46 that are screened, add homology arm sequences at both ends of the expression cassette and construct infectious clones, and transfect CEF cells. As shown in Table 2, the above 5 promoters can not be rescued to obtain recombinant viruses at 3 foreign gene insertion sites as the expression original of gD protein. It is shown that after the above promoters are combined with gD protein into an expression cassette, they cannot be compatible with the SC9-1 genome well, and recombinant viruses cannot be obtained. The infectious clones constructed by the above conventional method are not infective, and can not successfully obtain viruses that can be passed down after transfection of cells, so further optimization schemes are needed.

[0041] Table 2 Screening results of different promoters of gD (ILTV) protein expression cassette

[0042]

[0043]

[0044] Furthermore, the present invention screened a TK promoter, and used the screened ILTV's own TK promoter as the promoter element of the gD protein, and used HSV TK polyA as the terminator to construct a gD gene expression cassette marked as TK promoter-gD-PolyA. This gD protein expression cassette system has not been reported in the development of ILTV-related vector vaccines published publicly at home and abroad.

[0045] In order to illustrate the effect of the above-mentioned gD protein expression cassette system, the present invention also uses other expression cassette systems in the published patent (CN116648259A) as a control, that is, the gD gene promoter and the gI gene promoter are used as the promoter elements of the gD protein and the gI protein, respectively, and their own terminators are used as the protein expression termination sequence. The above-mentioned patent uses serum type III Marek's disease turkey herpes virus (HVT) as a vector to express the gD / gI protein of ILTV, which can provide good protection for ILTV, but the protection effect for the strong strain of serum type I Marek's virus may be insufficient. In the present invention, by introducing the gD / gI gene expression cassette disclosed in the above-mentioned patent as a control, it is marked as gD / gI promoter-gD / gI-PolyA to study whether it can achieve similar or better effects on the prevention and control of ILTV or MDV in the SC9-1 vector, thereby forming a comparative description.

[0046] The three sites of the screened SC9-1 genome were then used as candidate insertion sites for exogenous gene expression cassettes, namely, the expression cassettes TK promoter-gD-PolyA and gD / gI promoter-gD / gI-PolyA were inserted into the UL3 / 4, UL41 and UL45 / 46 sites respectively. A total of 6 vector vaccine strains were constructed, and their biological characteristics and immune protection efficacy in animals were studied.

[0047] Example 3 Construction of transfer vector

[0048] By PCR and gene recombination technology, the TK promoter of ILTV, the gD gene of ILTV, the HSV TK PolyA and the Kana expression cassette were cloned into the vector pBluescript II KS(+ / -) to construct the plasmid pBlu-TK promoter-gD-PolyA-Kana. By the same method, the gD / gI promoter of ILTV, the gD / gI gene of ILTV, the gD / gI terminator of ILTV and the Kana expression cassette were cloned into the vector pBluescript II KS(+ / -) to construct the plasmid pBlu-gD / gI promoter-gD / gI-PolyA-Kana.

[0049] Primers were designed based on the ILTV genome sequence isolated and preserved in the laboratory to amplify the TK promoter, gD gene, gD / gI promoter and gD / gI terminator respectively; primers were designed based on the gene sequence of the pK13 plasmid to amplify the Kana expression cassette sequence; based on the gene sequence of the vector pBluescript II KS(+ / -), the homology arm gene sequence used to construct the transfer vector was synthesized on the primer; based on the gene sequence of the vector pAcGFP1-N1, the HSV TK PolyA gene sequence was directly synthesized on the primer. Primer synthesis was completed by Shanghai Shenggong Biotechnology Co., Ltd., and the primer sequences are shown in Table 3.

[0050] Table 3 Primers for construction of transfer vector

[0051]

[0052] Note: The underlined part represents the homology arm sequence used for gene assembly when constructing the transfer vector.

[0053] The genomic DNA of ILTV was extracted as a template, and the TK promoter sequence and gD gene were amplified using primers TK-F / TK-R and gD-F1 / gD-R1 respectively; the Kana expression cassette gene was amplified using primers Kana-F1 / Kana-R1 with plasmid pK13 as a template. The vector pBluescript II KS(+ / -) was double-digested with Spe I / Kpn I, and then subjected to agarose gel electrophoresis together with the above three PCR amplified fragments, and the positive bands were recovered. 2.5ul of each of the recovered four gene fragment products were mixed and quantified according to the method. Instructions for High-Fidelity DNA Assembly Master Mix Add 10ul HiFi DNA Assembly Master Mix, mix well and incubate at 50℃ for 1h before transforming competent E. coli cells JM109. Pick a single colony for PCR identification, using TK-F / Kana-R1 as the primer. After the positive clone is sequenced correctly, save it and name the constructed plasmid pBlu-TKpromoter-gD-PolyA-Kana. Figure 1 shown.

[0054] Using ILTV genomic DNA as a template, primers gD-UP1 / gD-UP2, gD-F2 / gD-R2, gD-Down1 / gD-Down2 were used to amplify the gD / gI promoter, gD / gI gene, and gD / gI terminator sequence; using plasmid pK13 as a template, primers Kana-F2 / Kana-R2 were used to amplify the Kana expression cassette gene. The vector pBluescript II KS (+ / -) was double-digested with Spe I / Kpn I, and then subjected to agarose gel electrophoresis together with the above 4 PCR amplified fragments, and the positive bands were recovered. 2ul of each of the recovered 5 gene fragment products were mixed and quantified according to the method. Instructions for High-Fidelity DNA Assembly Master Mix Add 10ul HiFi DNAAssembly Master Mix, mix well and incubate at 50℃ for 1h before transforming competent E. coli cells JM109. Pick a single colony for PCR identification, using primers gD-UP1 / Kana-R2. After the positive clone is sequenced correctly, save it and the constructed plasmid is named pBlu-gD / gI promoter-gD / gI-PolyA-Kana. Figure 1 shown.

[0055] Example 4 Construction of infectious clone plasmid and rescue of recombinant virus

[0056] 4.1 Construction of infectious clone plasmid

[0057] According to the gene sequence of the constructed transfer vector and the genome sequence of SC9-1, the infectious clone plasmid construction primers and identification primers were designed to insert the TK promoter-gD-PolyA-Kana expression cassette and the gD / gI promoter-gD / gI-PolyA-Kana expression cassette into the UL3 / 4 site, UL41 site and UL45 / 46 site of the SC9-1 genome, respectively. Primer synthesis was completed by Shanghai Shenggong Biotechnology Co., Ltd., and the primer sequences are shown in Table 4.

[0058] Table 4 Primers for construction and identification of infectious clones

[0059]

[0060] Note: Lowercase letters represent homology arm sequences used at different insertion sites when constructing infectious clone plasmids.

[0061] Chloramphenicol (Cm + ) resistant LB culture plate to resuscitate the E. coli engineered bacteria EL250 containing the SC9-1 genome. Pick a single colony to contain Cm + The culture was inoculated into LB liquid medium at 32°C and 220 rpm overnight. The bacterial solution was inoculated into 100 mL of Cm + In the LB liquid medium with antibiotics, after shaking and culturing at 32℃ and 220rpm for 2-3h, when the OD600 value reaches about 0.6, the bacterial solution is placed in a 42℃ water bath for 20min to induce the expression of the recombinase protein in EL250. Immediately put the bacterial solution in an ice bath for 30min, pour it into a 50ml centrifuge tube and centrifuge it at 4℃ and 4000rpm for 10min, collect the bacteria, and pour out the supernatant until all the bacterial solution is processed. Add 20ml pre-cooled autoclaved 10% glycerol to gently resuspend the bacteria, mix well, and centrifuge horizontally at 4℃ and 4000rpm for 10min to collect the bacteria; repeat the above operation twice again. Use 500ul pre-cooled autoclaved 10% glycerol to gently resuspend the bacteria and distribute them in 1.5mL sterile centrifuge tubes, 50ul per tube, and store them at 4℃.

[0062] Using plasmid pBlu-TK promoter-gD-PolyA-Kana as template, primers TK-F-UL3 / 4, TK-R-UL3 / 4, TK-F-UL41, TK-R-UL41 and TK-F-UL45 / 46, TK-R-UL45 / 46 were used for PCR amplification. After agarose gel electrophoresis, the positive bands were recovered as electrotransformation fragments. Take 500ng of the recovered gene fragments and add them to an EL250 competent cell containing the SC9-1 genome and mix gently, transfer to a 1mm electrotransformation cup, electroshock under the conditions of 2000V, 100Ω, 25uF, quickly add to 1ml of LB liquid culture medium, mix well, transfer to a test tube and add 1ml of culture medium, and culture at 32℃, 180rpm with shaking for 2-3h. Take 300ul of bacterial solution and spread it on the Cm + The cells were plated on LB plates with Kana resistance and cultured overnight in a 32°C incubator. Single colonies were picked and identified by PCR using primers UL3 / 4-F, UL3 / 4-R, UL41-F, UL41-R, and UL45 / 46-F, UL45 / 46-R. The positive colonies were named SC9-TK-Kana (UL3 / 4), SC9-TK-Kana (UL41), and SC9-TK-Kana (UL45 / 46). Figure 2 shown.

[0063] For the positive bacteria identified above, 50ul of bacterial solution was inoculated into 5mL of LB liquid culture medium containing Cm antibiotics at a ratio of 1:100, and cultured in a 32℃ constant temperature shaking incubator at 220rpm for 2-4h. When the OD600 value reached about 0.4-0.6, L-arabinose with a final concentration of 1% was added for induction, and the Kana resistance gene was removed by recombination in vivo. After culturing in a 32℃ constant temperature shaking incubator for 1 hour, the bacterial solution was appropriately diluted and spread on a plate containing Cm + The cells were cultured overnight at 32°C in a resistant LB plate. Single colonies were picked for PCR identification using primers UL3 / 4-F, UL3 / 4-R, UL41-F, UL41-R, and UL45 / 46-F, UL45 / 46-R. The positive colonies were named SC9-TK (UL3 / 4), SC9-TK (UL41), and SC9-TK (UL45 / 46) as infectious clone plasmid strains for transfection. Figure 2 shown.

[0064] The same method was used to construct the gD / gI promoter-mediated infectious cloning plasmid. The primers used for electroporation were gD-F-UL3 / 4, gD-R-UL3 / 4, gD-F-UL41, gD-R-UL41, gD-F-UL45 / 46, gD-R-UL45 / 46, and the template was pBlu-gD / gI promoter-gD / gI-PolyA-Kana. The identification primers were UL3 / 4-F, UL3 / 4-R, UL41-F, UL41-R, and UL45 / 46-F, UL45 / 46-R. The obtained infectious clone plasmid strains containing kanamycin resistance were named SC9-gD-Kana (UL3 / 4), SC9-gD-Kana (UL41) and SC9-gD-Kana (UL45 / 46); the infectious clone plasmid strains without kanamycin resistance were named SC9-gD (UL3 / 4), SC9-gD (UL41) and SC9-gD (UL45 / 46). Figure 2 shown.

[0065] 4.2 Rescue of recombinant viruses

[0066] Extract infectious clone plasmids SC9-TK (UL3 / 4), SC9-TK (UL41), SC9-TK (UL45 / 46) and SC9-gD (UL3 / 4), SC9-gD (UL41), SC9-gD (UL45 / 46) by alkaline lysis method, and measure DNA plasmid concentration. Prepare chicken embryo fibroblasts (CEF), prepare secondary CEF cells 2-4 hours before transfection, spread 6-well cell culture plates, and culture under 37°C, 5% CO2 conditions. Change to fresh M199 culture medium containing 4% fetal bovine serum before transfection, transfect according to the requirements of the calcium phosphate transfection kit instructions, and transfect 10ug of infectious clone plasmid in each well. After transfection, continue to culture for 5 days under 37°C, 5% CO2 conditions to observe cell pathological changes. If no pathological changes are observed, continue to observe after blind passage of CEF cells for one generation until recombinant virus is obtained. The 6 recombinant viruses obtained were named as follows: TK promoter: SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), SC9-gD (TK, UL45 / 46); gD / gI promoter: SC9-gD (gD / gI, UL3 / 4), SC9-gD (gD / gI, UL41), SC9-gD (gD / gI, UL45 / 46).

[0067] Example 5 Identification of genetic stability of recombinant viruses

[0068] The six recombinant viruses constructed were serially passaged on CEF cells for 20 generations, and DNA was extracted from the 1st, 5th, 10th, 15th and 20th generations of recombinant viruses. Among them, SC9-gD (TK, UL3 / 4) and SC9-gD (gD / gI, UL3 / 4) were identified by PCR and sequenced using primers UL3 / 4-F and UL3 / 4-R; SC9-gD (TK, UL41) and SC9-gD (gD / gI, UL41) were identified by PCR and sequenced using primers UL41-F and UL41-R; SC9-gD (TK, UL45 / 46) and SC9-gD (gD / gI, UL45 / 46) were identified by PCR and sequenced using primers UL45 / 46-F and UL45 / 46-R. The results are shown in Table 1. Figure 3 , Figure 4 As shown, all the strains of each generation tested could amplify positive bands and were sequenced correctly, without any gene deletion, insertion or recombination, indicating that all six recombinant strains could be stably inherited.

[0069] Example 6 Evaluation of the growth kinetics of the recombinant SC9-1 vector vaccine strain in CEF cells

[0070] The 6 recombinant viruses SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), SC9-gD (TK, UL45 / 46), SC9-gD (gD / gI, UL3 / 4), SC9-gD (gD / gI, UL41), SC9-gD (gD / gI, UL45 / 46) and the parent strain SC9-1 strain preserved in the laboratory were inoculated in a 6-well cell culture plate covered with CEF cells at a dose of 100 PFU / well, and the recombinant viruses were collected every 24 hours after infection until 144h. To ensure the accuracy of the data, 3 replicate wells were taken for parallel experiments at each time point for each sample, and the average value was taken when calculating the data. The number of virus plaques (PFU) of each sample at each time point was measured, and a growth curve was drawn to compare the proliferation ability of each strain.

[0071] The results are as follows Figure 5As shown, the proliferation curves of SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), and SC9-gD (TK, UL45 / 46) are basically consistent with the SC9-1 parent strain, and only SC9-gD (TK, UL41) shows a slight decrease; while the proliferation abilities of SC9-gD (gD / gI, UL3 / 4), SC9-gD (gD / gI, UL41), and SC9-gD (gD / gI, UL45 / 46) all show varying degrees of decrease; all strains reach the peak of proliferation at around 144h. This indicates that the expression of ILTV gD protein by the TK promoter does not affect the replication of the recombinant virus, while the insertion of the gD / gI expression cassette system into the SC9-1 genome reduces the replication ability of the virus and affects the proliferation of the virus.

[0072] Example 7 Indirect immunofluorescence (IFA) assay to identify protein expression

[0073] The six recombinant virus strains SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), SC9-gD (TK, UL45 / 46), SC9-gD (gD / gI, UL3 / 4), SC9-gD (gD / gI, UL41), SC9-gD (gD / gI, UL45 / 46) and the parental strain SC9-1 were diluted and inoculated into a 96-well cell culture plate filled with CEF cells and cultured at 37°C and 5% CO2 until typical plaques appeared on the cells. The culture medium was discarded, the cells were washed three times with PBS, 100ul of cold anhydrous ethanol was added to each well for fixation, and the cells were fixed in a 4°C refrigerator for 2h; the fixative was discarded, the cells were washed three times with PBST, 100ul of primary antibody (ILTV polyclonal antibody serum, 1:100 dilution) was added to each well, and after incubation at room temperature for 1h, the cells were washed three times with PBST, and 100ul of FITC-labeled secondary antibody (rabbit anti-chicken, 1:500 dilution) was added to each well; the cells were incubated at room temperature in the dark for 30min, the liquid was discarded, the cells were washed three times with PBST, and the protein expression was observed under a fluorescence microscope.

[0074] The results are as follows Figure 6 As shown, all six recombinant viruses can express the target protein, and the fluorescence displayed by the SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), and SC9-gD (TK, UL45 / 46) groups is stronger. This shows that the exogenous gene expression cassette is inserted into the three sites of UL3 / 4, UL41, and UL45 / 46 of the SC9-1 genome, and the use of the TK promoter and the gD / gI promoter can both promote protein expression well, and the TK promoter has a stronger ability to promote protein expression. No fluorescence was seen in the SC9-1 inoculation group and the blank control group.

[0075] Example 8 Evaluation of the immune protection of recombinant viruses against ILTV virulent attack in SPF chickens

[0076] 90 one-day-old SPF chickens were randomly divided into 9 groups, each with 10 chickens. Groups 1-6 were immunized with recombinant viruses SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), SC9-gD (TK, UL45 / 46), SC9-gD (gD / gI, UL3 / 4), SC9-gD (gD / gI, UL41), and SC9-gD (gD / gI, UL45 / 46), respectively. Group 7 was immunized with parental strain SC9-1, and group 8 was immunized with commercial ILTV live vaccine (K317 strain). Group 9 was inoculated with PBS as a control group. Groups 1-7 were immunized with 3000 PFU / 0.2 ml of the corresponding virus strains by subcutaneous inoculation in the neck at 1 day of age, while group 8 was inoculated with 10 ILTV live vaccine vaccines by eye drops and nasal drops. 2.7 EID 50 / 0.1ml dose of K317 vaccine strain, and the control group in group 9 was subcutaneously inoculated with 0.2ml PBS in the neck. The specific grouping and immunization method are shown in Table 5.

[0077] After immunization, the chickens were isolated and kept, and the status of the chicks was observed every day for 14 consecutive days. Blood was collected 14 days, 21 days and 28 days after immunization, and the antibody production level was detected using the ILT antibody detection kit produced by BioChek. The antibody-positive samples were judged according to the standards established by the kit, and the antibody-positive rate was calculated. 28 days after immunization, all chickens were challenged with the ILTV virulent Wanggang strain by intratracheal injection, and each chicken was challenged with 10 4.0 EID 50 / 0.2ml, observe for 10 days.

[0078] Table 5 SPF chicks immunization and virus attack grouping

[0079]

[0080]

[0081] During the 14 days of observation after immunization, the chicks in groups 1-7 did not show any abnormal conditions such as illness, depression, lameness, etc., just like the control group in group 9. Their feeding and drinking were normal. However, the chicks in group 8 immunized with K317 live vaccine began to show abnormal conditions on the third day after immunization, showing symptoms such as eye inflammation, tearing, coughing, and respiratory rales. Generally, they began to return to normal 2-5 days after the onset of symptoms. No chickens died. During the entire observation period, a total of 7 chicks became ill to varying degrees, and 3 were normal. This shows that the SC9-1 strain and its recombinant vector vaccine strain are safe for chicks, while the K317 vaccine strain has a certain toxicity to 1-day-old chicks and is not suitable for immunization of young chicks.

[0082] By detecting the serum antibodies of chicks 14 days, 21 days and 28 days after immunization, the results are shown in Table 6. The use of TK promoter can better induce the production of antibody levels. The antibody positivity rate of the TK promoter immunization group (Groups 1-3) is similar to that of the K317 live vaccine immunization group (Group 8), and is significantly better than that of the gD promoter immunization group (Groups 4-6); the antibody positivity rate of the 1-3 immunization groups continued to increase from 14 days to 28 days, while the K317 immunization group began to decrease slightly on the 28th day, indicating that the immune persistence of SC9-1 vector vaccines SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL41), and SC9-gD (TK, UL45 / 46) is better than that of the K317 live vaccine group; SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL4 5 / 46) and K317 immunization groups could achieve 100% protection rate against the attack of ILTV strong strains, followed by SC9-gD (TK, UL41), while the protection rates of SC9-gD (gD / gI, UL3 / 4), SC9-gD (gD / gI, UL41) and SC9-gD (gD / gI, UL45 / 46) could not reach 80% or above, which could not meet the requirements of vaccine candidate strains for vaccine protection efficiency; after immunization of chicks with SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL45 / 46) and K317 vaccines, the antibodies against ILTV produced, although not 100% of the chick serum samples could turn positive, still provided 100% protection against virus attack, indicating that cellular immunity also played an important role.

[0083] Table 6 Protection of each immune group of SPF chickens after ILTV challenge

[0084]

[0085] Example 9 Evaluation of the immune protection of recombinant viruses against ILTV virulent challenge in commercial chickens

[0086] One-day-old commercial chicks were randomly divided into 5 groups, 10 in each group. The 4 immune groups were immunized with SC9-gD (TK, UL3 / 4), SC9-gD (TK, UL45 / 46), K317 and SC9-1, respectively, and the other group was immunized with PBS as a control. The specific immunization method and dosage, the challenge method and dosage are the same as those shown in Table 5 in Example 8. Among them, the commercial chicks were blooded once before immunization (1 day old) and 28 days after immunization (28 days old) to measure antibodies against ILTV. The observation method after immunization and challenge is the same as in Example 8.

[0087] After continuous observation for 14 days after immunization, 2 chicks in the K317 immunization group showed mild eye inflammation symptoms, but they returned to normal within 3 days, and the rest of the chicks did not show any abnormalities. Before immunization, the maternal antibody positive rate of commercial chicks was between 30% and 50%; 28 days after immunization, the antibody positive rates of SC9-gD (TK, UL3 / 4) and SC9-gD (TK, UL45 / 46) were 90% and 80%, respectively, both higher than the K317 immunization group (40% positive rate). At the same time, SC9-gD (TK, UL3 / 4) and SC9-gD (TK, UL45 / 46) immunization were less interfered by maternal antibodies and could provide 100% protection for commercial chicks; while the K317 live vaccine was interfered by maternal antibodies, resulting in a protection efficiency reduced to 80%. The results are shown in Table 7.

[0088] Table 7 Protection of commercial chickens in each immune group after ILTV challenge

[0089]

[0090]

[0091] Example 10 Evaluation of the immune protection of recombinant viruses against MDV

[0092] Two recombinant virus strains, SC9-gD (TK, UL3 / 4) and SC9-gD (TK, UL45 / 46), which can provide complete protection against ILTV, as well as the parental strain SC9-1 and the control strain HVT FC-126 were selected as evaluation objects to assess their protective efficiency against the virulent MDV strain Md5.

[0093] 50 one-day-old SPF chickens were randomly divided into 5 groups, 10 in each group. There were 4 immunization groups and 1 control group. The first 4 groups were subcutaneously inoculated with SC9-gD (TK, UL3 / 4) strain, SC9-gD (TK, UL45 / 46) strain, SC9-1 strain and HVT FC-126 strain, 3000 PFU / 0.2ml / chicken, respectively, and the 5th group was subcutaneously injected with 0.2ml PBS in the neck. On the 6th day after immunization, all chicks were intraperitoneally injected with the super-virulent MDV strain Md5, 1000 PFU / 0.2ml / chicken. The incidence of chickens in each group was observed every day, and the incidence and mortality of each group were counted. After 13 weeks, all experimental chickens were dissected and the lesions were observed. For the immunization groups that did not develop the disease, 5 chickens were randomly selected from each group to collect the internal organs of the heart, liver, spleen, lungs, and kidneys to make pathological sections and observe the pathological changes; while all the internal organs of the non-dead chickens in the control group were taken to make pathological sections.

[0094] The results are shown in Table 8. The chickens in the SC9-gD (TK, UL3 / 4) strain, SC9-gD (TK, UL45 / 46) strain and SC9-1 strain immunization groups all showed normal symptoms without any clinical symptoms. However, the chickens in the control group became ill and died 2 weeks after the infection. The chickens with the disease showed stunted development, slow growth, ruffled feathers, depression, and obvious paralysis symptoms. The peak of death occurred about 5 weeks after the infection, and only one chicken survived by the end of the experiment. One chicken in the HVT FC-126 strain immunization group became ill in the 4th week after the infection, showing leg paralysis and obvious weight loss, but no death occurred during the observation period, and no clinical symptoms were observed in the remaining chickens. Anatomical and pathological observations showed that the internal organs of chickens in the SC9-gD (TK, UL3 / 4) strain, SC9-gD (TK, UL45 / 46) strain and SC9-1 strain immunization groups were normal, and no lesions were observed; three chickens in the HVT FC-126 strain immunization group had similar pathological changes as one surviving chicken in the control group, which showed obvious lymphocyte nodules in the spleen, and microscopic lesion observations showed focal lymphocyte-like cells of different sizes in the kidneys, with equal nuclear divisions. This shows that the live vector vaccine has the same immunogenicity as the parent strain, and can provide 100% protection against MDV super-virulent; while the immune protection effect of HVT FC126 is limited, with a protection efficiency of only 70%.

[0095] Table 8 Protection of each immune group of SPF chickens after MDV challenge

[0096]

[0097] Note: Those with clinical symptoms and pathological changes in internal organs are recorded as onset.

[0098] Combined with the above results, it can be seen that the recombinant MDV vector vaccine strains SC9-gD (TK, UL3 / 4) and SC9-gD (TK, UL45 / 46) obtained by the screening of the present invention have good in vitro proliferation characteristics, stable genome genetics, and can stably express gD protein; there are no side effects or pathogenic effects after immunization of SPF chickens and commercial chickens, and both can provide 100% protection against ILTV virulent attack; immunization of SPF chickens can provide 100% protection against MDV virulent attack.

[0099] It is known that, regarding HVT-ILT vector vaccine, Merck Animal Health has an HVT-ILT vector vaccine that has been marketed, but relevant data show that it cannot provide 100% protection against ILTV. HVT vector vaccine is also mentioned in CN 118086231A, but it cannot provide 100% protection against ILTV. At the same time, the animal evaluation results in the present invention also show that HVT cannot provide 100% protection against MDV super-toxicity. At present, the fowlpox virus live vector vaccine FPV-ILT, which is available on the market both at home and abroad, is also used for the prevention and control of ILTV. Although it can avoid the side effects and virulence reversion of ILTV live vaccine, it is easily affected by fowlpox virus maternal antibodies, the expression amount of exogenous proteins is also limited, and the duration is short. The MDV (SC9-1 strain) vector vaccine of the present invention is not only safe, but also can reduce the interference of maternal antibodies to the greatest extent, one-time vaccination can provide lifelong immunity, and the long duration of immunity can be used to develop other vector vaccines, and the application prospect is broad.

Claims

1. A recombinant Marek's virus vector vaccine strain expressing the gD protein of infectious laryngotracheitis virus of avian chickens, characterized in that: A gD gene expression cassette was constructed using TK promoter as the promoter element of the gD protein of infectious laryngotracheitis virus and HSV TK polyA as the terminator. A recombinant Marek's virus vector vaccine strain expressing the gD protein of infectious laryngotracheitis virus of chickens was constructed by inserting the gD gene expression cassette into the UL3 / 4 or UL45 / 46 site of the type I MDV SC9-1 genome used as a vector.

2. The recombinant Marek's virus vector vaccine strain according to claim 1, characterized in that: The nucleotide sequence of the TK promoter is shown in SEQ ID NO: 1, the nucleotide sequence of the HSV TK polyA is shown in SEQ ID NO: 2, and the nucleotide sequence of the gD gene is shown in SEQ ID NO:

3.

3. The recombinant Marek's virus vector vaccine strain according to claim 1, characterized in that: The amino acid sequence of the gD protein is shown in SEQ ID NO:

4.

4. The recombinant Marek's virus vector vaccine strain according to any one of claims 1 to 3, characterized in that: The recombinant Marek's virus vector vaccine strain is a live attenuated vector vaccine.

5. Use of the recombinant Marek's virus vector vaccine strain according to any one of claims 1 to 4 in the preparation of a medicament for simultaneous challenge and protection against infectious laryngotracheitis virus and Marek's disease virus.

6. Use of the recombinant Marek's virus vector vaccine strain according to any one of claims 1 to 4 in the preparation of a medicament for preventing animals from being simultaneously infected with infectious laryngotracheitis virus and Marek's disease virus.

7. The use according to claim 5 or 6, characterized in that The recombinant Marek's virus vector vaccine strain is administered subcutaneously in the neck, and the immunization dose is 3000 PFU / 0.2 ml.

8. The use according to claim 7, characterized in that 28 days after immunization, the chickens were challenged with the highly virulent Wanggang strain of infectious laryngotracheitis virus by intratracheal injection. 4.0 EID 50 / 0.2ml, the protection rate reaches 100%.

9. The use according to claim 7, characterized in that: Six days after immunization, the chickens were challenged with the highly virulent strain of Marek's virus Md5 by intraperitoneal injection, with 1000 PFU / 0.2 ml per chicken, and the protection rate reached 100%.

10. DIVA vaccine, characterized in that It is prepared using the recombinant Marek's virus vector vaccine strain described in any one of claims 1-4.

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