文献类型：期刊文献

中文题名：尼罗罗非鱼NCCRP-1基因的原核表达及条件优化

英文题名：Prokaryotic Expression and Condition Optimization of NCCRP-1 Gene in Nile tilapia

作者：黄瑜[1,2];牛金中[1,2];汤菊芬[1,2];鲁义善[1,2];汪志文[1,2];郑琦[1,2];蔡佳[1,2];简纪常[1,2]

机构：[1]广东海洋大学水产学院,湛江524088;[2]广东省水产经济动物病原生物学及流行病学重点实验室,湛江524088

年份：2018

卷号：37

期号：9

起止页码：3753

中文期刊名：基因组学与应用生物学

外文期刊名：Genomics and Applied Biology

收录：CSTPCD、、北大核心2017、CSCD2017_2018、北大核心、CSCD

基金：广东海洋大学博士学位论文培育项目(201602);国家自然科学基金(31572651)共同资助

语种：中文

中文关键词：尼罗罗非鱼;NCCRP-1;原核表达;Western;blotting

外文关键词：Oreochromis niloticus;NCCRP-1;Prokaryotic expression;Western blotting

中文摘要：非特异性细胞毒性细胞受体蛋白（non-specific cytotoxic cell receptor protein 1,NCCRP-1）是一种在鱼类非特异性细胞毒性细胞（non-specific cytotoxic cell,NCC）活化过程起到关键作用的受体蛋白,也是NCC表面的一种分子标记。本研究根据NCBI上已公布的罗非鱼NCCRP-1基因序列,设计一对带酶切位点的引物,经PCR扩增、酶切、连接等步骤,构建罗非鱼NCCRP-1基因的原核表达质粒p GEX-NCCRP-1,将重组质粒导入至大肠杆菌BL21中,成功表达了NCCRP-1重组蛋白。对影响NCCRP-1表达的IPTG浓度、诱导时间、诱导温度等因素进行了优化,发现在20℃下,采用0.2 mmo I/L IPTG诱导5 h,可以诱导NCCRP-1可溶性重组蛋白的高效表达。而免疫印迹结果显示,经纯化后的重组蛋白可与GST-Tag单克隆抗体发生特异性反应,进而表明表达的重组蛋白是罗非鱼NCCRP-1蛋白。该结果为后续罗非鱼NCCRP-1的单克隆抗体制备以及NCCRP-1相关的功能研究奠定了重要基础。

外文摘要：Non-specific cytotoxic cell receptor protein 1（NCCRP-1） is a receptor protein that plays a key role in the activation of non-specific cytotoxic cells（NCC）, and it is also a molecular marker on the surface of NCC. In this study, a pair of primers with enzyme digestion sites were designed based on the tilapia NCCRP-1 gene sequence published on NCBI. The prokaryotic expression plasmid p GEX-NCCRP-1 of the tilapia NCCRP-1 gene was constructed by PCR amplification, digestion and ligation. The recombinant plasmid p GEX-NCCRP-1 was introduced into E. coli BL21 and the NCCRP-1 recombinant protein was successfully expressed. The effects of IPTG concentration, induction time and induction temperature on the expression of NCCRP-1 were optimized. It was found that the high expression of NCCRP-1 soluble recombinant protein was induced with 0.2 mmol/L IPTG at 20℃ for 5 h. The western blot results showed that the purified recombinant protein could react specifically with the GST-Tag monoclonal antibody, which further indicated that the expressed recombin ant protein was tilapia NCCRP-1 protein. This result would provide an important basis for the preparation of monoclonal antibody and related function research of tilapia NCCRP-1.