PIWIL4的克隆及其抗体制备和初步应用

目的 制备兔抗人PIWIL4的多克隆抗体,鉴定其特异性,并应用该抗体检测内源性PIWIL4在人各细胞系中的表达差异及细胞定位.方法 构建原核表达质粒pGEX-5X-1-PIWIL4,转化大肠杆菌BL21,PIWIL4蛋白经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.融合蛋白通过切胶纯化后免疫家兔制备抗体血清.以间接酶联免疫吸附试验(ELISA)检测抗体效价,Western blot鉴定抗体特异性及检测PIWILA在各细胞系中的表达差异,免疫荧光染色观察PIWIL4的细胞定位.结果 成功构建原核表达质粒,表达并纯化PIWIL4蛋白.免疫大白兔后得到PIWIL4多克隆抗体,ELISA检测抗体效价为1:20 000,Western blot和免疫组化确定抗体具有高度特异性,并成功地应用该抗体检测到PIWIL4在人多种细胞系中的表达差异及细胞定位.结论 PIWIL4蛋白及其多克隆抗体的成功制备,为进一步研究PIWILA的生物学功能奠定了基础.

Gene cloning and polyclonal antibody preparation of human PIWI4 and preliminary application

Objective To generate rabbit polyclonal antibody against human PIWIL4 protein, to identify its functional characterization, measure differential expression and determine the cellular localization of PIWIL4 protein in various cell lines. Methods Prokaryotic expressed plasmid pGEX-5X-1-PIWIL4 was constructed and transformed to E. coli BL21 to induce expression by IPTG. The fusion protein was injected into rabbits subcutaneously to produce polyclonal antibodies after purification by gel regaining. Enzyme linked im-munosorbent assay (ELISA) was operated to detect the titer of the antibodies, western blotting was used to identify the specificity and sensitivity of the antibodies and detect the differential expression of PIWIL4 protein in various cell lines. Meanwhile, immunofluorescence experiments were used to show cellular localization of PIWIL4 protein. Results The prokaryotic expressed plasmid was constructed correctly. PIWIL4 protein was expressed and purified, and then rabbit polyclonal antibodies against PIWIL4 were generated after immunization with the fusion protein. The titer detected by ELISA was 1:20 000. The evidence of western blotting demonstrated the specificity of the antibodies. Finally, we successfully observed the differential expression and cellular localization of PIWIL4 protein in various cell lines. Conclusion The polyclonal antibody against PIWIL4 protein has been achieved successfully. It will be propitious for the intensive functional study of PIWIL4.