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

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.