蛋鸡TRPV6基因的原核表达、纯化及抗体制备

Prokaryotic expression,purification and polyclonal antibody preparation of TRPV6 gene in laying hen

为制备蛋鸡TRPV6蛋白多克隆抗体,根据其基因序列,设计1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1 801～2 176nt的375bp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,试验成功构建原核表达载体pET-32a(+)-TRPV6,SDS-PAGE蛋白电泳检测发现目的蛋白大小约35 000;Western blot分析显示,表达的TRPV6融合蛋白具有良好的免疫原性,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∶100 000。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具有重要作用。

To prepare the polyclonal antibody of TRPV6 protein in laying hen, a pair of primers was designed and syn- thesized to clone the 1 801 to 2 176 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 BL2I （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 pre pared antibody. The results showed that the prokaryotic expression vector pET-32a （ ＋）-TRPV6 was successfully constructed in this experiment and the relative molecular weight of the clone recombinant protein was 35 000 detec- ted with gel electrophoresis polyacrylamide. Western blot indicated that the TRPV6 fused protein displayed great im- munobiological activity and the purified anti-TRPV6 polyclonal antibody could bind with TRPV6 protein which was specially expressed in E. coli. The titer of antiserum generated was 1 - 100 000 by ELISA; Therefore, the TRPV6 fused protein and polyclonal antibody obtained could play an important role in studying the function and mechanism of TRPV6 calcium ion channel in the formation of medullary bone in laying hen.