Nogo-66蛋白氨基酸肽的克隆及在原核细胞的表达

Cloning and expression of nogo-66 protein in prokaryotic cells

目的:克隆编码Nogo-66氨基酸多肽基因,构建原核重组表达载体,并诱导其在大肠埃希菌BL21(DE3)中表达。方法:逆转录聚合酶链反应(RT-PCR)方法扩增出编码Nogo蛋白环外-66氨基酸多肽基因,将其克隆于T载体PMD18-T,酶切鉴定后再亚克隆于原核表达载体pET-42a(+),酶切及测序证实序列正确后,转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D半乳糖苷(IPTG)诱导表达融合蛋白-Nogo-66。结果:克隆了编码Nogo蛋白环外-66氨基酸多肽基因,构建了融合蛋白的重组表达质粒,融合蛋白的表达随着时间延长增加。结论:获得了编码Nogo蛋白环外-66氨基酸多肽基因及其原核表达产物,对研究Nogo蛋白的生物学功能及其mAb的制备奠定基础。

Objective： To clone Nogo-66 gene, construct the recombinant prokaryotic expressive vector and express its products in E. coll. Methods： Nogo-66 gene was amplified by reverse transcription polymerase chain reaction （RT-PCR） and then cloned into PMD18-T vector. After identified by the enzyme digestion, the gene was subcloned into prokaryotic expressive vector pET-42a（ ＋ ）, which was identified by the enzyme digestion and sequencing. Nogo-66 fusion protein was expressed in E. coli BL21 （DE3） by I PTG. Results：The Nogo-66 gene and recombinant prokaryotic expression vector were obtained and the Nogo-66 protein was expressed and increased with prolong of time. Conclusions： The Nogo-66 expression vector and its prokaryotic expression product were obtained. It maybe has a great significance for study the biologic function of Nogo-66 protein and the preparation of monoclonal antibody against Nogo-66 protein.