摘要
根据已发表的番鸭细小病毒(MDPV)核苷酸序列设计并合成1对引物,对MDPV非结构蛋白NS2基因进行扩增。所获得的PCR产物与预期片段大小相符,约1.4 kb。将该片段直接与pMD18-T载体连接,转化入感受态大肠杆菌DH5α中增殖。在对所提质粒进行快速鉴定、PCR扩增及BamHI酶切线性化初步鉴定的基础上,经限制性内切酶BamHI和SalI进行双酶切鉴定。将克隆产物pMD18-T-NS2与原核表达载体pET-32a分别用BamHI和SalI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BamHI单酶切、BamHI和SalI双酶切,证实含有目的基因,且基因插入方向正确,成功构建了含有MDPV NS2非结构蛋白基因的原核表达载体,为高效原核表达及研制更为有效的基因工程疫苗打下了基础。
In this study,a pair of primers were designed according to the published DNA sequence of MDPV and was used to amplify the NS2 gene of MDPV by polymerase chain reaction（PCR）.The PCR product was then cloned into the vector pMD18-T.In order to construct a prokaryotic expression vector containing the NS2 gene of MDPV,double digestion（BamH I and Sal I）were performed to deal with pMD18-T and pET-32a respectively for the directional ligation of the two fragments.The following restriction endonuclease analysis has proved the correctness of the expression vector.Construction of the prokaryotic expression vector has laid the foundation of the expression of NS2 protein and the development genetically engineering vaccine.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第4期88-90,共3页
China Animal Husbandry & Veterinary Medicine
