稳定表达猪水疱病病毒P1基因的PK-15细胞系的建立  

Establishment of a Stable PK-15 Cell Line Expressing the P1 Gene of Swine Vesicular Disease Virus

作　　者：田宏[1] 吴锦艳[1] 龚真莉[1] 郑海学[1] 孙世琪[1] 尚佑军[1] 刘湘涛[1] 谢庆阁[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《畜牧兽医学报》2008年第4期478-482,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家863高技术研究发展计划(2003AA241110)

摘　　要：从猪水疱病全长感染性cDNA中,应用PCR技术扩增到SVDV结构蛋白P1基因,并在目的片段的5′端引入Kozak序列(Kozak,1987),定向克隆于逆转录病毒载体pBABE puro。经PCR、酶切和序列分析鉴定,获得阳性重组质粒。将该重组质粒与水疱性口炎病毒载体pVSV-G共转染GP2-293细胞,收获假型病毒,在Polybrene的介导下感染PK-15细胞,嘌呤霉素筛选阳性细胞克隆。免疫荧光连续检测阳性克隆传代细胞,发现在不同代次的细胞中均有SVDV P1蛋白表达,而且表达的蛋白可被SVD阳性血清所识别;同时应用PCR技术,可从体外反复传代阳性细胞基因组中扩增到SVD P1基因。表明本次所筛选的阳性细胞克隆不但能持续稳定地表达SVD P1蛋白,而且可携带外源基因进行传代,具有良好的遗传稳定性。P1 gene of swine vesicular disease virus （SVDV） was amplified by PCR using specific primers from SVDV HK/70 genome. The amplified fragment was cloned into pBABE puro vector for sequence analysis,and the recombinant plasmid was named pBABE puro-P1. Then pBABE puro-P1 and pVSV-G were cotransfected into GP2-293 packaging cells by liposomes, and recombinant retrovirus was acquired. The recombinant retroviruses was transfected into PK-15 cell by polybrene. The transfectants were selected by paromycin. Results of immunofluorescence, PCR analysis and Western blot showed that the foreign gene was integrated into the chromosome of transfected PK-15 cells and the expressed protein could react with positive serum against SVDV.

关 键 词：猪水疱病病毒P1基因 重组逆转录病毒载体 PK-15细胞 基因表达 

分 类 号：Q813[生物学—生物工程]