转录靶向猪Jiv基因shRNA细胞株的建立 及抗猪瘟病毒转基因猪的构建

【目的】构建转入靶向猪Jiv基因shRNA干扰片段的阳性细胞株，通过比较各细胞株对猪瘟病毒增殖的干扰效果，筛选对猪瘟病毒增殖有明显抑制作用的细胞株，为抗猪瘟转基因猪的构建提供材料。【方法】研究设计了靶向猪Jiv基因的4个shRNA干扰片段，并构建插入干扰片段的慢病毒(P1、P2、P3、P4)。将慢病毒分别转染PK-15细胞，阳性细胞接种猪瘟病毒后72 h用实时荧光定量PCR检测猪瘟病毒RNA的量，以比较4种干扰细胞株对猪瘟病毒增殖的干扰效果。将对猪瘟病毒增殖有较好干扰效果的P2慢病毒干扰载体转染猪胎儿成纤维细胞，获得稳定表达靶向猪Jiv基因shRNA干扰片段的猪胎儿成纤维细胞株，作为抗猪瘟病毒转基因猪构建的供体细胞，将细胞核移植到成熟的去核猪卵母细胞中，获得体细胞核移植胚胎，移植入受体母猪输卵管中进行转基因猪构建，对获得的转基因猪进行外源基因的鉴定。【结果】获得了4株转入靶向猪Jiv基因shRNA干扰片段的PK-15细胞株，其中转入P2干扰载体的细胞株对猪瘟病毒的增殖有明显的抑制作用，将转入P2干扰载体的猪胎儿成纤维细胞株为核供体细胞通过体细胞核移植，获得经鉴定为外源基因插入阳性的转基因猪。【结论】细胞Jiv基因的表达对猪瘟病毒的增殖有一定的影响，筛选获得的一个干扰细胞株对猪瘟病毒的增殖有明显的干扰效果；通过体细胞核移植技术获得一头转入靶向猪Jiv基因shRNA干扰片段（P2）的转基因猪。

Establishment of Cell Lines Transcribing shRNA Targeted to Jiv Gene and Constuction of CSFV Resistant Transgenic Piglet

【Objective】To construct postive cell strans inserted the shRNA fragments targeting to porcine J-Domain protein interacting with virus gene, the interference effects were evaluated by comparing the proliferation of classical swine fever virus (CSFV) in constructed cell strains and the cell strain with significant CSFV resistance would be provided as the basic materials for constructing the CSFV-resistant transgenic pig. 【Method】 Four shRNA interfering fragments were designed tagerting to pig Jiv gene and 4 lentivirus inserted the designed shRNA fragment（named P1, P2, P3 and P4）. Four positive PK-15 cell strains transfected with lentivirus were established individually and the interference effects of CSFV were evaluated by determining the RNA of CSFV in positive cell strains after 72 h infected with CSFV by real-time RT-PCR . The porcine fetus fibroblasts were isolated and infected with lentivirus P2 which interfered the proliferation of CSFV remarkerably in cell. A positive porcine fetus fibroblast strain stable expressing the shRNA interfering fragment targeting to Jiv gene was established which also was the donor cells for constructing the transgenic pig. The nuclear of positive pircine fetus fibroblast was transferred into matured enucleated porcine oocytes, and somatic embryos were obtained and transplanted into the receptor sows for constructing transgenic pig. The foreign gene was identified in obtained transgenic pig.【Result】 Four PK-15 cell strains inserted into the shRNA interfering fragments targeting to the Jiv gene of porcine were established, the P2 cell strain (inserted into P2 interfering fragment) was significantly CSFV-resistant. The nuclear of porcine fetus fibrlblast inserted P2 interfering fragment was donor cells for constructing transgenic pig by somatic cell cloning and embryo transplantation, and a transgenic piglet was obtained.【Conclusion】 The expression of Jiv gene can influence the proliferation of CSFV in cells. A PK-15 cell strain with stable expressing shRNA P2 was established which could resist CSFV proliferation remarkablly. A trnasgenic piglet was constructed by somatic cell cloning and embryo transplantation which carries the P2 interfering fragment targeting to porcine Jiv gene.