大鼠寡霉素敏感相关蛋白(OSCP)基因的改造及在大肠杆菌中的表达

Modification of Rat OSCP Gene and Expression of OSCP in E.coli

目的通过将发生点突变的两个大鼠寡霉素敏感相关蛋白(OSCP)基因进行改造,获得具有正确序列的OSCP基因,在大肠杆菌中表达并进行活性鉴定。方法通过限制性核酸内切酶和T4DNA连接酶将发生错义突变的两个OSCP基因的DNA克隆进行改造,获得具有正确核苷酸序列的OSCP基因;然后将该片段连接进原核表达载体pET-28c(+),构建成重组质粒PET-28c(+)-OSCP;用该重组质粒转化E.coli,转化子在37℃诱导表达相应的融合蛋白,通过Western-blot鉴定。结果酶切及测序显示对OSCP基因错义突变的纠正获得成功。酶切结果显示成功构建了原核表达载体pET-28c(+)-OSCP。IPTG诱导表达4h后,SDS-PAGE及Western-blot显示诱导表达出分子量约23000的大鼠寡霉素敏感相关蛋白,且该蛋白具有免疫活性。结论在国内初次表达出大鼠OSCP,表达产物具有免疫反应性,为OSCP的结构及功能研究奠定了基础。

Objective To obtain a corrected sequence of rat oligomycin sensitivity-conferring protein （OSCP） gene by modifying the two mutated rat OSCP genes which have point mutation,then express the OSCP gene in E.coli. Methods Modification was achieved by digesting the mutated OSCP genes with restriction endonuclease and followed by ligation with a correct nucleotide sequence using T4DNA ligase. The gene was then cloned into a prokaryotic expression vector pET-28e （＋） to construct a recombinant plasmid PET-28e （＋）- OSCP. E.coli were transformed with the recombinant plasmid and the transformants were induced to express at 37℃.IPTG-induced expression product was identified by SDS-PAGE and Western-blot. Results Digestion with restriction endonuelease and sequencing showed that the modification of the OSCP gene by missense mutation was successful. Digestion of the constructs with restriction endonuelease confirmed the construction of expression vector pET-28e（＋）-OSCP.SDS-PAGE and Western-blot analysis showed that the expression product showed immunoreactivity to antibody against OSCP and had a molecular weight of 23 kDa. Conclusion Immunoreaetive OSCP was successfully expressed in E.coli. The expression of OSCP protein may be used in the future study of structure and function of OSCP.