[0020] The invention discloses a highly efficient method for constructing a general vector for genetic engineering of avian type I Marek's virus vaccine strain and its application in constructing recombinant Marek's disease virus for vaccine production.
[0021] The present invention discloses a reporter and screening gene for constructing a general vector for genetic engineering of an efficient avian type I MDV vaccine strain. The gene is a green fluorescence and neomycin gene (S-g-n) designed and synthesized by the inventor, under the regulation of the SV40 early promoter High-efficiency fusion expression of a reporter and screening gene as a universal vector, such as the nucleotide sequence shown in sequence 1.
[0022] In the present invention, the non-essential region US10 for MDV replication is selected as the foreign gene insertion site, and at the same time, the optimal length of the homology arm of the universal vector is screened. The left and right lengths are respectively between 1000 and 2000 bp. nucleotide sequence. Connect the amplified homology arm gene (U) into the T plasmid (T is the plasmid pMD18-T, which is a product of TAKARA company, rebuilt from the pUC18 plasmid, and is a special plasmid for cloning genes), and construct a universal vector with the same Source arm plasmid (T-U).
[0023] The present invention newly synthesizes and transforms a multiple cloning site, introduces a suitable spacer, and constructs an expression cassette (C-M-A), such as the nucleotide sequence shown in Sequence 9.
[0024] The universal vector (T-Us) was constructed after linking the above elements.
[0025] Co-transfect with avian type I MDV to obtain the recombinant virus to verify the effectiveness of the vector.
[0026] In the present invention, the enhanced green fluorescent gene (derived from the pEGFP-C1 carrier, obtained through amplification and transformation) and neomycin gene (derived from the pCDNA3.1 (+) plasmid, obtained through amplification and transformation) are jointly used as avian type I MDV The reporter and screening genes of the general vector for genetic engineering of vaccine strains are regulated by independent early promoters and efficiently fused and expressed. The recombinant virus obtained in this way can not only be used for drug screening, but also convenient and intuitive detection of virus titer and content. In order to amplify the above-mentioned reporter screening gene (S-g-n), the present invention has designed and synthesized the following corresponding primers:
[0027] Sequence 2 (G1): 5'-GTGGGTCGACTAGTTATTAATAGTAATC-3';
[0028] Sequence 3 (G2): 5'-TGAGATCTACGCGTTAAGATACATTG-3';
[0029] Sequence 4 (N1): 5'-GGATCCATGATTGAACAAGATGGA-3';
[0030] Sequence 5 (N2): 5'-CTCGAGTCAGAGAGAACTCGTCAAG-3';
[0031] The construction process is as follows: the enhanced green fluorescent gene amplified by PCR with primers G1/G2 is digested with restriction endonucleases BglII and SalI, and recovered; the neomycin amplified by PCR with primers N1/N2 After the gene is cut with restriction endonucleases BamHI and XhoI, the recovery (because the gene can produce complementary sequences after digestion with BglII and BamHI, XhoI and SalI respectively, so it can be connected with each other) and the enhanced green fluorescence recovered before After the genes are ligated, they are used as PCR templates and amplified with primers G1 and G2 to obtain the reporter and screening genes (S-g-n) of the universal vector.
[0032]The invention analyzes the genome structure and function of the poultry type I Marek virus, screens non-essential regions for virus replication, and selects the US10 gene in the genome as the insertion site of the exogenous gene on the basis of analyzing these non-essential regions. This can not only ensure that the replication of the avian I-type Marek virus is not affected, but also ensure the efficient and stable expression of the foreign gene, and enhance the genetic stability of the recombinant Marek virus. The present invention selects the sequences on both sides of the US10 gene as the homology arm (U) of the universal vector for the genetic engineering of the poultry type I Marek virus vaccine strain, and designs and synthesizes a pair of PCR primers, preferably sequence 7 (M1) and sequence 8 (M2). After amplifying the primers showing the sequence, the amplified product was connected to the T vector (T is the vector pMD18-T, which is a product of TAKARA company, rebuilt from the pUC18 vector, and is a special vector for cloning genes) while constructing a universal vector. Source arm plasmid (T-U).
[0033] The following primers were designed and synthesized to amplify the homology arm (U) of the universal vector:
[0034] Sequence 7 (M1): 5'-GAGTATTGTGAAGCACT-3';
[0035] Sequence 8 (M2): 5'-GAAACTGGTCAGTGGAG-3';
[0036] The invention adopts the method of genetic engineering to construct the expression cassette (C-M-A) of the genetic engineering universal vector of the poultry type I Marek virus vaccine strain. The specific process is as follows: design a pair of PCR primers, preferably the primers of sequences shown in sequence 9 (C1) and sequence 10 (C2) to amplify the product obtained, which contains cytomegalovirus (CMV) early promoter (Promoter), multiple The cloning site (MCS) and the transcription termination signal polyA gene are introduced downstream of the SalI restriction site; at the same time, an appropriate spacer (IR) is selected and connected to the multiple cloning site (MCS) after being digested by EcoRV to construct an expression Cassette (C-M-A), so as to ensure the simultaneous high-efficiency expression of multiple foreign genes under the control of one promoter.
[0037] The following PCR primers were designed and synthesized for amplifying the expression cassette of the universal vector, such as sequence 10 (C1) and sequence 11 (C2):
[0038] Sequence 10 (C 1 ): 5'-GTTCTGCTGGTTGACATTGATTATTGA-3';
[0039] Sequence 11 (C2): 5'-TAGTCGACCCATAGAGCCCACCGC-3';
[0040] Construct universal carrier (T-Us) after the nucleotide sequence connection of avian I type MDV vaccine strain genetic engineering general carrier as mentioned above, concrete process is as follows:
[0041] The PCR-amplified reporter gene and screening gene (S-g-n) shown in sequence 1 and the PCR-amplified expression cassette gene (C-M-A) shown in sequence 9 were respectively digested with restriction endonuclease SalI, Recovery, in vitro ligation as a PCR template, using the above primers G1 and G2, using the Taq enzyme that can produce blunt ends, amplified by PCR to obtain the gene sequence (with blunt end), the reporter gene, screening gene (S-g-n), and expression cassette gene (C-M-A) were recovered; the restriction endonuclease PstI was used to digest the two ends and the two ends were filled with DNA polymerase I to obtain the homologous arm gene plasmid ( T-U) (T is the carrier pMD18-T, which is a product of TAKARA company, rebuilt from the pUC18 vector, and is a special carrier for cloning genes), and finally connect the reporter gene with the screening gene (S-g-n) and the expression cassette gene (C-M-A) The homologous arm gene plasmid (T-U) was inserted, and the ligated product was transformed into Escherichia coli to obtain the general engineering vector (T-Us) of the avian type I Marek virus vaccine strain.
[0042] The recombinant method of bird I type MDV is as follows:
[0043] The universal vector (T-Us) plasmid was transfected into chicken embryo fibroblasts infected with CVI988. After the virus plaques appeared, the green fluorescent virus plaques were observed under a fluorescent microscope, and the results indicated that the exogenous genes had been recombined into the Marek virus genome. After drug screening for 5 generations (CEF cells pressurized with 0.4mg/ml G418 culture medium for 24h, inoculate the recombinant virus, continue to culture at 37°C, replace 50% of the culture medium every 24h to maintain the G418 screening pressure constant, in the 5th The infected cells were digested, and then inoculated on fresh cells, and repeated 4 times) to obtain the purified avian type I recombinant MDV CVI988-26 strain (or Gallid herpesvirus 2 CVI988-26, June 18, 2009 This virus strain has been sent to the General Microorganism Center of China Microbiological Culture Collection Management Committee for preservation, and the preservation number is: CGMCCNo.3132).
[0044] The above results show that the genetically engineered universal carrier (T-Us) of the avian I-type Marek virus vaccine strain constructed by the present invention can regulate the high-efficiency expression of foreign genes, has the characteristics of high efficiency, quickness and stability, and overcomes the shortcomings of existing transfer vectors.
[0045] Positive significance of the present invention
[0046] The invention constructs a universal carrier (T-Us) of avian I-type Marek virus, only needs several generations of drug screening and fluorescence screening, and realizes the design goal of stably and efficiently expressing foreign gene recombinant virus in a short period of time. Simultaneously, the present invention also provides a set of techniques for screening and purifying recombinant Marek's virus, and obtained a strain of recombinant avian I-type Marek's disease virus CVI988-26 strain, which carries the universal vector T- Us, immunizing animals with this virus can not be affected by maternal antibodies, stimulate the body to produce antibodies against various viruses, and can effectively protect birds from the attack of avian Marek virus. It has laid a solid foundation for the development of a new type of Marek's disease vaccine.
