摘要
为克隆南阳牛BoLA-DRA基因,构建原核表达载体并研究该基因在大肠埃希菌中的表达。从南阳牛脾脏组织中提取总RNA,利用RT-PCR方法扩增得到BoLA-DRA基因,将其克隆至pGEM-Teasy克隆载体上,转化感受态细胞DH5α,经测序鉴定后,进一步亚克隆至原核表达载体pGEX-4T-1中,构建出重组表达质粒,经IPTG诱导表达。结果表明,本试验成功克隆了大小为917 bp的BoLA-DRA基因,重组质粒pGEX-4T-DRA在大肠埃希菌中以包涵体形式表达,表达产物经SDS-PAGE和Western blot鉴定大小为54.4 ku,与预期结果一致。为进一步研究该蛋白的功能及制备抗体奠定了基础。
This study aimed to clone Nanyang cattle BoLA-DRA gene, construct recombinant prokaryotic expression vector and to study the gene expression in Escherichia coll. Firstly, BoLA-DRA gene fragment was amplified by RT-PCR after total RNA was extracted from spleen of Nanyang cattle. Following, the BoLA-DRA gene was cloned into the pGEM-T vector and transferred into E. coli DHSa. After sequence a- nalysis, the BoLA-DRA gene was cloned into prokaryotic expression vector pGEX-4T-1 and induced by IPTG （isopropyl β-D-l-thiogalactopyranoside）. Results showed that the 917 bp long BoLA-DRA gene was cloned, the fusion protein was obtained successfully,and it existed mostly in the form of inclusion body. I-dentification of expressed products by SDS-PAGE and Western blot showed that the target protein was 54.4 ku which was consistent with the expected results. The results laid a foundation for further studying on the function of the protein and preparation of antibody.
出处
《动物医学进展》
CSCD
北大核心
2010年第6期5-10,共6页
Progress In Veterinary Medicine
基金
国家十一五科技支撑计划项目(2008BADB2B03-03)
国家863计划项目(2008AA101010)
