Establishing method of bacterial artificial chromosome recombinant duck plague virus rescue system platform and application
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An artificial chromosome and duck plague virus technology, applied in the field of veterinary medicine, can solve the problems of unknown gene function, genetic manipulation and difficulty in constructing recombinant virus
Inactive Publication Date: 2016-07-27
SICHUAN AGRI UNIV
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[0003] Duck plague virus Chinese virulent strain (DPVCHv), as a member of the herpes virus family, has a huge genome and complex genome structure, with as many as 76 coding genes, making it particularly difficult to genetically manipulate and construct recombinant viruses
Due to the constraints of the research platform, the pathogenic mechanism of duck plague virus
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Embodiment 1
[0061] Example 1 Bacterial Artificial Chromosome Recombination Duck Plague Virus Rescue System Platform Construction
[0062] 1. Primer design: According to the TK gene and its left and right gene sequences, pEGFP-C1 sequence and pBeloBAC11 sequence, the primers used to construct the bacterial artificial chromosome recombinant duck plague virus rescue platform are designed as shown in Table 1:
[0063] Table 1 Primers used in the construction of bacterial artificial chromosome recombinant duck plague virus rescue platform
[0064]
[0065]
[0066] 2 Construction of bacterial artificial chromosome recombinant duck plague virus transfer vector
[0067] The insertion site of exogenous BAC sequence in constructing bacterial artificial chromosome recombination duck plague virus rescue system platform is the TK region of DPVCHv. Design and construct a bacterial artificial chromosome transfer vector containing left and right homologous arms of TK gene, and obtain recombinant ...
Embodiment 2
[0191] Example 2 Construction of recombinant duck plague virus UL55 gene deletion strain and its in vitro biological characteristics
[0192] 1 strain / plasmid
[0193] The maps of plasmid strains pKD46, pKD4, and pCP20 used for Red recombination are as follows: Figure 7 shown.
[0194] 2 Primer design
[0195] The primers used to construct the UL55 gene deletion and its reversion mutant strains in this example are shown in Table 6.
[0196] Table 6 is used for the related primers of RED recombination
[0197]
[0198] 3 The first round of RED recombination to knock out the UL55 gene
[0199] 3.1 Amplification and recovery of UL55 gene homologous targeting fragment
[0200] The UL55 sequence to be knocked out is replaced by the targeting fragment of the kanamycin resistance gene locus with UL55 gene homology arms and FRT sequences on both sides. Using the plasmid pKD4 as a template, the kanamycin resistance gene was amplified. The 20 bases at the 3' end of the primer...
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Abstract
The invention discloses an establishing method of a bacterial artificial chromosome recombinant duck plague virus rescue system platform and application of the platform. A bacterial artificial chromosome recombinant duck plague virus is obtained by inserting a recombinant duck plague virus transfer vector pUC18/EGFP-TKAB-BAC11 in a TK domain, wherein the recombinant duck plague virus transfer vector contains a TK gene left-right homologous arm, a reporter gene EGFP and a bacterial artificial chromosome core function component. By means of the platform, the in-vitro biologics characteristics of a UL55 gene-deleted strain established through an inside-bacterium two-step RED recombination method and a back mutation strain and parent strain of the UL55 gene-deleted strain are quite close; the functions are not related to positioning of a UL26.5 gene in a cell. The method is beneficial to development of pathogenic mechanism and gene function research of DPV CHv and is beneficial to the duck plague virus prevention and the research and application of recombinant duck plague virus vaccines of other poultry infectious diseases based on the platform; in addition, due to the fact that the recombinant virus carries a TK deletion mark and an EGFP gene, a mark vaccine can be developed to clinically distinguish a wild virus and a recombinant vaccine virus.
Description
technical field [0001] The application belongs to the field of veterinary medicine, and in particular relates to a construction method and application of a bacterial artificial chromosome recombination duck plague virus rescue system platform. Background technique [0002] Duck plague (Duck Plague), also known as duck virus enteritis (Duckviral enteritis, DVE), is commonly known as "big head plague" in my country, and is an acute disease of ducks, geese, swans, geese and other Anseriformes caused by duck plague virus (Duck Plague Virus). , febrile, septic, contact infectious diseases. It is characterized by vascular injury, tissue hemorrhage, rash-like lesions in certain parts of the gastrointestinal mucosa, specific lesions in lymphoid organs, and degenerative changes in parenchymal organs. The clinical manifestations of sick ducks are high fever, green and thin feces, numb feet, tears, and swelling of the head and neck of some sick ducks; esophageal mucosa is bleeding, oft...
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