蛋鸡TRPV6基因的原核表达、 纯化及抗体制备.docVIP

  蛋鸡 TRPV6 基因的原核表达、 纯化及抗体制备# 郑燕玲1，侯加法2** 5 10 15 20 25 30 35 40 （1. 南京农业大学动物医学院，畜禽骨骼生物学实验室，南京 210095； 2. 南京农业大学动物医学院，南京 210095） 摘要：为制备蛋鸡 TRPV6 蛋白多克隆抗体，根据基因序列，设计一对特异性引物，以卵巢组 织中提取的总 RNA 为模板，扩增蛋鸡 TRPV6 基因 1801 至 2176 位置的 375 bp 序列，构建原 核表达质粒 pET-32a(+)-TRPV6；将重组质粒转化 BL21（DE3），经 IPTG 诱导表达 TRPV6 融 合蛋白，通过镍离子螯合柱纯化后免疫新西兰大白兔，获得兔抗鸡 TRPV6 多克隆抗体，分别 通过酶联免疫吸附试验（ELISA）法和 Western-blot 检测抗体的效价和抗体特异性。结果表 明，本试验成功地构建原核表达载体 pET-32a(+)-TRPV6，SDS蛋白电泳检测发现目的 蛋白约 35 kD；Western-blot 分析显示，表达的 TRPV6 融合蛋白具有良好的免疫源性，其抗 体可与大肠杆菌表达的产物特异性结合；ELISA 显示其抗体效价达 1:100000。因此，获得纯 化的融合蛋白和多克隆抗体对研究 TRPV6 钙离子通道在蛋鸡髓质骨形成的作用机理起到重 要作用。 关键词：鸡；TRPV6；原核表达；纯化；多克隆抗体 中图分类号：请 S854 Prokaryotic expression，purification and polyclonal antibody preparation of TRPV6 gene in laying hen Zheng Yanling1, Hou Jiafa2 (1. Laboratory of Bone Biology, College of Veterinary Medicine, Nanjing Agricultural University, NanJing 210095; 2. College of Veterinary Medicine, Nanjing Agricultural University, NanJing 210095) Abstract: To prepare the polyclonal antibody of TRPV6 protein in laying hen, a pair of primers was designed and synthesized to clone the 1801 to 2176 position of TRPV6 gene about 375 bp by RT-PCR from the total RNA, which was extracted from ovarium. The amplicated segment was inserted to the expression plasmid pET-32a(+), then the prokaryotic expression vector pET-32a(+)-TRPV6 was constructed and transformed into BL21(DE3) and induced by IPTG. The recombination proteins were purified by using Ni2+-NTA chelating column. The purified proteins were inoculated into New Zealand adult rabbits to develop polyclonal antibody of rabbit against chicken TRPV6. Enzyme-linked immunoadsorption assay (ELISA) and Western-blot were then performed to evaluate the specificity of the prepared antibody. The results showed that the prokaryotic expression vector pET-32a(+)-TRPV6 was successfully constructed in this experiment and the clone recombinant protein relative molecular weight was 35 kD detected with gel electrophoresis polyacrylamide. Weste