摘要
目的在大肠杆菌系统表达并纯化人精子肌动蛋白样蛋白7a(ACTL7a)的N段(1-70),制备兔抗ACTL7a多克隆抗体。方法构建重组表达质粒pGEX-6p-1-ACTL7a(1-70),以重组质粒转化E.coli BL21(DE3),筛选阳性重组菌,IPTG诱导目的蛋白表达,通过镍离子金属螯合树脂和谷胱甘肽琼脂糖凝胶4B树脂两次亲和纯化和分子筛分离目的蛋白;以表达的ACTL7a(1-70)蛋白免疫新西兰大白兔,制备抗ACTL7a的多克隆抗体。ELISA检测抗体效价,Western blot法鉴定抗体特异性,免疫荧光组织化学技术检测ACTL7a在曲精小管细胞的表达。结果经IPTG诱导,在大肠杆菌中表达出目的蛋白ACTL7a(1-70),纯化后免疫新西兰大白兔,成功获得抗血清。血清效价达到1:160000以上,且具有良好的特异性。结论成功构建了ACTL7a基因的原核表达载体,并在大肠杆菌中诱导表达,经纯化获得高纯度的目的蛋白;制备出兔抗ACTL7a抗体,效价及特异性均良好。
Objective To express and purify the recombinant human sperm actin-like protein 7a （ACTL？a） N terminal （1-70） in E. coli, and prepare the corresponding rabbit anti-ACTLTa polyclonal antibodies. Methods We constructed the recombinant expression plasmid pGEX-6p-I-ACTLTa （1-70）, transformed the plasmid into E. coli BL21 （DE3）, and selected positive recombinant strain. The target protein was induced to express by IPTG and purified by nickel ions-chelating resin and glutathione sepharose 4B resin, and finally obtained by molecular sieve chromatography. The purified ACTLTa （1-70） protein were used to immunize New Zealand rabbits to prepare anti-ACTLTa polyclonal antibodies. The antibody titers were detected by ELISA, the antibody specificity by Western blotting, and the localization of ACTLTa in the spermatogenic cells of seminifer- ous tubule epithelium by immunofluorescence histochemistry. Results The target protein ACTL？a （1-70） was successfully expressed by IPTG in E. coli, and the corresponding antiserum was obtained in the rabbits immunized with it. The titer of the antiserum reached 1：160 000 and it was proved with a good specificity. Conclusion ACTLTa was expressed in the constructed prokaryotic expression vector. After purification and immunization, high titer and specific anti-ACTL？a antibody was prepared in rabbits.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第5期507-510,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家科技支撑计划(2012BAI31B08)
国家重点实验室专项基金(2060204)
