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Construction and Cell Transfection of the Targeting Vectors of Jiv90 Genes in Swine

Author: LiYuLi
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus Jiv90 gene Targeting vector RNA interference
CLC: S858.28
Type: Master's thesis
Year: 2011
Downloads: 35
Quote: 0
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Abstract


Classical swine fever (Classic swine fever, CSF) is a highly contagious disease. Classical swine fever virus (Classical swine fever virus, CSFV) is enveloped single-stranded positive-strand RNA virus, is the Flaviviridae (Flaviviridae) pestivirus (Pestivirus) an important member. CSFV can infect a variety of cells, including T cells, high and low density granulocytes, monocytes, peripheral blood leukocytes, macrophages and dendritic cells. Jiv chaperone host cell gene (J-domain protein interacting with viral protein) are present in the host cell of a CSFV infection in the continuous role. In addition, studies have also found that the 3'-UTR CSFV virus infection plays an important role. First, according to Jiv90 genes and CSFV infection in this study to take effective gene targeting and the recent positive and negative selection (PNS) gene targeting strategy, according to the Cre-Loxp system homologous recombination, gene construct pig Jiv90 homologous targeting vector, and transfected porcine umbilical vein endothelial cells (Swine Umbilical Vein Endothelial cells SUVEC) knockout effect for Jiv90 preliminary validation. Secondly, classical swine fever virus 3'-UTR of the viral infection played an important role in the design on the region targeted three sites shRNA sequences, using RNA interference method further given pathogenesis of CSFV research. In this study, the following results were obtained: (1) published in GenBank Jiv90 primers designed to extract SUVEC whole genome DNA, cloned Jiv90 gene sequence on both sides of the core area, as the length of the targeting vector homology arms. Arm length gene fragments were 4522 bp and 1944 bp. Homologous short arms were connected with the carrier pEASY-T1, the long arm through the Hind Ⅲ digestion and sequencing single, short arm after Cla Ⅰ and Spe Ⅰ double digestion and sequencing, are in line with replacement vector homology arms build requirements. (2) the length of the Jiv90 homologous gene targeting backbone vector pA2T arm and connected successfully constructed Jiv90 knockout vector JKS. Use of liposomal transfection reagent JKS vector was transfected SUVEC, through neomycin (G418) and ganciclovir (GANC) screened stably transfected cell lines. Wherein G418 screening concentration of 1500μg/mL, GANC of 200 nmol / mL. (3) the use of PCR detection of the neo gene in the cell genome integration situation, and after screening positive cells were infected with CSFV by real-time quantitative PCR (Real-time PCR) detection of the virus cell proliferation. The results showed that JKS vector transfected cells swine fever virus nucleic acid content than normal cells decreased 3.65 times, indicating Jiv90 gene knockout swine fever virus inhibit cell proliferation. (4) Design of three loci targeting shRNA sequences. ShRNA template synthesis and connected to pGPU6/GFP/Neo vector sequencing proved successfully constructed against CSFV 3'-UTR of the three loci interference carrier. Vectors through liposome transfected PK-15 and SIEC cells. G418-resistant screened by the interference vectors expressing more than 85% positive cells by Real-time PCR to determine Pig1 carrier interference with good results.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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