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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。
Abstract：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.
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基本信息:
DOI：10.13417/j.gab.037.003753
中图分类号:S917.4
引用信息:
[1]黄瑜,牛金中,汤菊芬,等.尼罗罗非鱼NCCRP-1基因的原核表达及条件优化[J].基因组学与应用生物学,2018,37(09):3753-3758.DOI:10.13417/j.gab.037.003753.
基金信息:
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
2017-09-22
2017-09-22
2017-09-22