河南华溪蟹生殖调控分子VASA的原核表达、抗体制备及免疫鉴定

Prokaryotic expression, antibody preparation and immunological identification of VASA protein in the freshwater crab(Sinopotamon henanense)

为了研究生殖调控分子VASA在河南华溪蟹性腺发育过程中的表达模式和功能,本研究制备了VASA蛋白特异的多克隆抗体。选取河南华溪蟹vasa基因813 bp的特异区段,克隆到p ET32a载体,构建了原核表达载体p ET32a-Shvasa,转化至大肠杆菌BL21,经IPTG诱导表达和SDS-PAGE检测。结果显示,47 ku的VASA融合蛋白在菌液上清液中大量存在。VASA融合蛋白经Ni-NTA His-Bind亲和层析柱分离纯化后,免疫新西兰大白兔,制备了河南华溪蟹VASA蛋白的多克隆抗体。ELISA检测显示,VASA蛋白多克隆抗体的效价高达1.0×105。进一步通过免疫吸附实验和Western blot方法鉴定抗体特异性,研究表明,获得的多克隆抗体不仅能识别VASA融合蛋白,而且能特异识别河南华溪蟹卵巢中的天然VASA蛋白。研究为鉴定河南华溪蟹及其他蟹类生殖细胞提供了有效手段,为进一步解析十足目动物VASA蛋白的功能提供了分子基础。

In order to study the expression pattern and function of the reproductive regulatory molecular VASA during gonadal development of the freshwater crab,Sinopotamon henanense（Sh VASA）,we have cloned the partial sequence of vasa gene in the freshwater crab,S.henanense（Shvasa）.Based on the sequence,in the present study,we prepared the specific anti-VASA polyclonal antibody for exploring the expression pattern and function of Sh VASA protein during gonadal development.A specific segment of 813 bp of Shvasa gene was selected and cloned into p ET32a vector to construct the prokaryotic expression vector p ET32a-Shvasa.After the recombinant vector was transferred into Escherichia coli,the recombinant VASA protein was expressed under the induction of IPTG.SDS-PAGE analysis showed that the fusion protein,which was about 47 ku,mainly existed in the supernatant.Using the fusion protein purified by Ni-NTA His-Bind affinity chromatography column as antigen,we immunized the New Zealand white rabbits,and obtained the polyclonal antibody of Sh VASA protein.The titer of anti-VASA polyclonal antibody reached 1×10^5 in ELISA assay.Furthermore,we identified the specificity of the antibody gained in this study.Immuno-adsorption and Western blot analysis indicated that the produced antiVASA polyclonal antibody could specifically bind to the fusion protein as well as the natural Sh VASA protein extracted from ovary.These results will provide a powerful tool for identification of germ cells in S.henanense and other crabs,and for the study on the function of VASA protein in decapoda.