Construction method of porcine epidemic diarrhea virus infectious clone
A porcine epidemic diarrhea and infectious cloning technology, applied in the field of bioengineering, can solve problems such as inability to effectively meet customer needs, no specific therapy, and weak protective effect
Pending Publication Date: 2022-07-29
新疆方牧生物科技有限公司
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Problems solved by technology
However, existing clinical data show that the vaccine produced by the original old strain G1 group (PEDV CV777 strain) has been unable to provide new variant strains (variant strains (G2 group)) of porcine epidemic diarrhea that have appeared on the market in recent years. adequate protection
3. Studies have found that mixed monoclonal antibodies and yolk antibodies have a weak protective effect, and interferon can reduce weight loss to a certain extent in pigs, but there is no specific treatment for the disease
Because porcine epidemic diarrhea virus is not easy to multiply in cells, the production effect of the vaccine has been unsatisfactory
Inactivated vaccines can only produce antibodies to prevent and control viruses, but cannot remove and eliminate viruses; live vaccines cannot effectively meet the needs of customers due to the use of traditional strain vaccines (PEDV CV777 strains), and customers urgently need a safe, Porcine Epidemic Diarrhea Vaccine Effectively Controlling Current Epidemic Strains
Method used
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[0063] A method for constructing an infectious clone of porcine epidemic diarrhea virus, comprising the following steps:
[0064] 1. Virus isolation:
[0065] Tissue disease materials: Select several healthy piglets from a farm in Hebei, and then infect the piglets with the porcine epidemic diarrhea G2 strain. After the piglets have diarrhea symptoms for 5 days, the jejunum intestinal tissue is taken;
[0066] Cells: Vero cells, prepared, identified and supplied by the laboratory;
[0067] Serum: Porcine epidemic diarrhea virus-specific positive serum and Porcine epidemic diarrhea negative serum were provided by China Veterinary Drug Administration.
[0068] Isolation of Porcine Epidemic Diarrhea Virus: The tissue samples were cut into pieces, homogenized, centrifuged at 3000 r / min for 5 minutes, the supernatant was filtered and sterilized with a 0.22 μm filter, inoculated with well-grown T-25 monolayer Vero cells, and incubated for 1 h Then, discard the virus solution, wash...
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The invention discloses a construction method of porcine epidemic diarrhea virus infectious clone, which comprises the following steps: uploading a porcine epidemic diarrhea G2 strain to a P3 generation in a Vero-CCL81 cell, and then identifying the isolated strain, including RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection, virus content detection and specificity test. The separated G2 type PEDV strain is subjected to genome sequence analysis, a specific amino acid mutation region in an S gene coding amino acid sequence of the G2 type PEDV strain is found out, a PEDV infectious clone receptor binding domain is replaced by an AscI restriction enzyme cutting site by utilizing a mature gene cloning and vector construction technology, and the G2 type PEDV infectious clone receptor binding domain is converted into an AscI restriction enzyme cutting site. Vero-CCL81 cells are transfected according to the extracted pBAC-PEDV (mut) plasmids and pBAC-PEDV positive control plasmids to carry out virus rescue, and a solid foundation is laid for the vaccine to enter target animal test and clinical test stages.
Description
technical field [0001] The invention relates to the field of bioengineering, in particular to a method for constructing an infectious clone of porcine epidemic diarrhea virus. Background technique [0002] Porcine Epidemic Diarrhea (PED) is a highly harmful contact intestinal infectious disease. The rapid spread of this disease has brought unprecedented challenges to the pig industry. The disease is caused by Porcine Epidemic Diarrhea Virus (PEDV) and is characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets. Pigs of different ages and breeds are susceptible, but The harm to suckling piglets, shelf pigs or fattening pigs is even greater, especially suckling piglets are the most seriously affected, with a morbidity rate as high as 100% and a mortality rate of 30% to 80%. In addition, although pigs of other ages are susceptible, the mortality rate tends to decrease with the increase of pig age. [0003] The prevention of porcine epidemic diar...
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Patent Timeline
Login to View More IPC IPC(8): C12N15/85C12N15/50C12N7/01A61K39/215A61P31/14
CPCC12N15/85C07K14/005C12N7/00A61K39/12A61P31/14C12N2770/20021C12N2770/20034C12N2770/20051C12N2800/107
Inventor 张满义梁军冯泽泰任新蓉蒲小峰
Owner 新疆方牧生物科技有限公司