小鼠脂多糖应答蛋白mlrpS的载体构建及融合蛋白表达

Construction of Expression Vector of mlrpS, A Mouse Protein of Lipopolysaccharide Response and Expression of mlrpS Fusion Protein

为构建小鼠mlrpS-cDNA基因原核、真核表达载体,大肠杆菌表达其融合蛋白。采用反转录-聚合酶链反应从经脂多糖刺激的鼠NIH3T3细胞cDNA中,扩增出编码mlrpS的cDNA。用限制性内切酶KpnⅠandXhoⅠ消化后,插入原核表达载体pTAT中,经酶切鉴定与测序证实后,转化大肠杆菌BL21(DE3)菌株。异丙基β-D硫代半乳糖苷(IPTG)诱导产生融合蛋白。经KpnⅠ和XhoⅠ酶切回收mlrpS-cDNA,插入pcDNA3.1载体中;pcDNA3.1-mlrpS再经KpnⅠ和ApaⅠ酶切后,插入到pEGFP-c1载体上,构建pEGFP-mlrpS融合的真核表达载体。构建mlrpS表达载体经测序证实,与GenBank登录的序列完全一致;双酶切鉴定证实,克隆基因正确插入载体pEGFP及pTAT;SDS-PAGE证实融合蛋白表达成功。说明:成功地构建了mlrpS原核、真核表达载体,成功正确表达了6His/mlrpS融合蛋白。

To construct a eukaryotic and prokaryotic expression vector of mlrpS and to express mlrpS fusion protein, the cDNA sequence encoding the mlrpS protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps （lipopolysaccharide）. The mlrpS-cDNA was digested by KpnⅠ and Xho Ⅰ and inserted into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT- mlrgS and pCDNA3.1-mlrpS. The protein was expressed in E. coli BL21 （DE3）. A fusion protein was expressed by IPTG induction, pCDNA3.1, mlrpS-cDNA was recovered by digested with KpnⅠ and Apa Ⅰ , and inserted into pEGFP-C1. The eukorytic expression vector containing mlrpS was indentical with the sequence submitted in GenBank and was accurately inserted into the vector pTAT, pCDNA3.1 and pEGFP which were confirmed by restriction endonucleases digestion. It is conclused that eukaryotic and prokaryotic expression vectors of mlrpS has been successfully constructed and 6His/mlrpS have been correctly expressed.