兔抗人精子顶体精子溶菌酶样蛋白1结合蛋白(SAS1B)多克隆抗体的制备及鉴定

Preparation and identification of rabbit anti-human sperm acrosome sperm lysozyme-like protein 1 binding protein(SAS1B)polyclonal antibody

目的制备兔抗人精子顶体精子溶菌酶样蛋白1结合蛋白(SAS1B)多克隆抗体。方法构建pET-22b-SAS1B-His质粒,转化E.coli.BL21(DE3)细胞,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导SAS1B重组蛋白表达。用层析纯化后的蛋白免疫日本大耳白兔制备SAS1B多克隆抗体。ELISA检测SAS1B抗体效价,Western blot和免疫组织化学法鉴定SAS1B抗体特异性。结果在构建pET-22b-SAS1B-His原核表达载体的基础上,在BL21(DE3)细胞成功诱导表达SAS1B重组蛋白。制备的兔抗人SAS1B多克隆抗体效价为1∶51200。SAS1B多克隆抗体能与SAS1B重组蛋白特异结合且能识别乳腺癌组织中的SAS1B蛋白。结论成功制备具有较好特异性的兔抗人SAS1B多克隆抗体。

Objective To prepare rabbit anti-human sperm acrosome sperm lysozyme-like protein 1 binding protein(SAS1B)polyclonal antibody.Methods The plasmid pET-22b-SAS1B-His was constructed and transformed into E.coli.BL21(DE3)cells.The expression of the recombinant SAS1B protein was induced by isopropyl-β-D-thiogalactoside(IPTG).The recombinant protein purified by affinity chromatography method was employed to immunize Japanese white rabbit for preparing SAS1B polyclonal antibody.The titer of the SAS1B antibodies was detected by ELISA and its specificity was identified by Western blotting and immunohistochemistry.Results Prokaryotic expression vector of pET-22b-SAS1B-His was successfully constructed,and recombinant SAS1B protein was efficiently expressed in E.coli BL21(DE3).The titer of the antibody was up to 1∶51200 as determined by ELISA.SAS1B polyclonal antibody could specifically bind recombinant SAS1B protein and recognize endogenous SAS1B protein in breast cancer tissue.Conclusion Rabbit anti-human SAS1B polyclonal antibody has been successfully prepared with good specificity.