鹅细小病毒主要结构蛋白基因的扩增、克隆与原核表达载体的构建

Cloning and Construction of a Protokaryotic Expressing Vector Containing the Main Structural Protein Gene of Goose Parvovirus

根据已发表的鹅细小病毒 ( GPV)核苷酸序列设计并合成了 1对引物 ,对 GPV主要结构蛋白 VP2和VP3基因进行扩增。所获得的 PCR产物与预期片段大小相符 ,约 1 .8kb。将该片段直接与真核表达 p CR3.1T载体连接 ,转化入感受态大肠杆菌 TOP1 0 F′中增殖。在对所提质粒进行快速鉴定、PCR扩增以及 Bam H 酶切线性化初步鉴定的基础上 ,经限制性内切酶 Sca 和 Eco R 分别酶切进行正反向鉴定 ,获得了 3个正向插入的克隆 ( PVPA)和 2个反向插入的克隆 ( PVPB)。将正向插入的克隆 PVPA1与原核表达载体 p ET-2 8b( + )分别用 Bam H 和 Xho 双酶切后 ,回收目的片段进行定向连接 ,并转化入感受态大肠杆菌 DH5α中。对所获得的重组质粒分别经 Sca 、Eco R 、Bam H / Xho 酶切 ,证实含有目的基因 ,且基因插入方向正确 ,成功地构建了含有 GPV主要结构蛋白基因的原核表达载体 。

Goose parvovirus(GPV) was first observed and isolated in 1956 by FANG Ding yi in Yang zhou,China,and it was proved to be the pathogen of a highly fatal disease of geese and muscovy ducks which was once called Derzsy′s disease.Little has been done with GPV′s molecular biology by now in China.In this study,primers P1 and P2 were designed according to the published DNA sequence of GPV and was used to amplify the main structural protein gene of GPV by polymerase chain reaction(PCR).The PCR product was then cloned into the eukaryotic expression T vector pCR3.1,and three direct recombinants(PVPA1 PVPA3) were obtained.In order to construct a prokaryotic expression vector containing the main structural protein gene of GPV,double digestion(BamHⅠ and XhoⅠ) were performed to deal with PVPA1 and pET 28b(+) respectively for the directional ligation of the two fragments.The following restriction endonuclease analysis has proved the correctness of the expression vector.