内皮型一氧化氮合酶运输诱导物NOSTRIN基因的克隆与表达

Study on clone and express endothelial nitric oxide synthase traffic inducer protein

目的 构成内皮型一氧化氮合酶运输介导物 (endothelialnitricoxidesynthasetrafficinducer,NOSTRIN)基因的原核表达重组体 ,并进行初步表达。方法 从人胎盘组织中提取总RNA ,经反转录 -聚合酶链式反应 (RT -PCR)扩增出NOSTRIN基因 ;将NOSTRIN基因克隆入 pGEM -T载体中 ,经DNA测序确认后 ,构建原核表达重组体 pRSET -NOS ,在工程菌BL2 1(DE3)中进行IPTG诱导表达。结果 所获得的NOSTRIN基因测序正确 ,并表达重组蛋白 ,经SDS -PAGE分析 ,相对分子量为 5 8KD。结论 构建的原核表达重组体 pRSET -NOS能高效表达重组NOSTRIN蛋白。

Objective: To construct prokaryotic expression recombinant of endothelial nitric oxide synthase traffic inducer and to express primarily recombinant NOSTRIN. Methods: Human NOSTRIN gene fragment was amplified from human placenta total RNA by RT-PCR, and cloned into the vector of pGEM-T. The sequence of NOSTRIN gene was analyzed. Prokaryotic expression vector pRSET-NOS was constructed and transformed into E.coli DH5 a. Recombinant NOSTRIN was expressed primarily in E.coli BL21(DE3) by IPTG induction. Results: The sequence of NOSTRIN gene obtained was correct. SDS-PAGE analysis indicated that expressed product was about 58KD. Conclusion: The recombinant of pRSET-NOS is an effective prokaryotic expression recombinant.