融合PCR技术在山羊痘病毒表达载体构建中的应用  

Application research on fusion PCR of construction of Goat pox virus expression vector

作　　者：宋书婷 张孝忠[1] 张秀萍[1,2] 陈宏伟[1,2] 李有文[1,2] 

机构地区：[1]塔里木大学动物科学学院,新疆阿拉尔843300 [2]新疆生产建设兵团塔里木畜牧科技重点实验室,新疆阿拉尔843300

出　　处：《黑龙江畜牧兽医》2017年第7期1-4,291,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30960020;31360616);塔里木畜牧科技兵团重点实验室开放课题(HS201307);塔里木大学研究生基金项目(TDGRI201604)

摘　　要：为了构建山羊痘病毒表达载体,试验利用融合PCR的方法连接4种组合的3个不同来源的基因片段,插入含有TK基因的载体中,快速构建羊痘病毒表达载体,并转染至BHK-21细胞中进行验证。结果表明:利用融合PCR技术成功融合了P11-7.5-ZSG、P11-7.5-GFP、I1L-7.5-ZSG、I1L-7.5-GFP 4个组合中的3个基因片段,产物长度均在1.2 kb左右,并插入PGEM-TK载体中,构建了4个表达载体,测序结果与理论上100%同源,转染BHK-21细胞可见绿色荧光。说明试验成功构建了4个山羊痘病毒的表达载体。A study was conducted to build goat pox virus expression vector. Three independent gene fragments were connected by the fusion PCR technology. The genes formed four combinations and inserted into the vectors, which contained TK gene, to build goat pox virus expression vector rapidly. And the BHK-21 cells were transfected by the vectors for verification. The results showed that Pll -7.5 -ZSG,P11 -7.5 - GFP 、 I1L - 7.5 - ZSG, I1 L - 7.5 - GFP were all composed of the three gene fragments successfully by fusion PCR technology, and the product length was about 1.2 kb. The products were inserted into PGEM - TK carrier, and four expression vectors were constructed,and consistent 100% homologous with sequencing results theoretically. The transfected BHK - 21 cells displayed green tluorescence. It was concluded that four Goat pox virus expression vectors were constructed successfully.

关 键 词：融合PCR 山羊痘病毒 表达载体 同源重组 载体构建 

分 类 号：S852.654[农业科学—基础兽医学]