主动免疫肌动蛋白样蛋白7a蛋白引起小鼠睾丸曲细精管的损伤  

Damage of Seminiferous Tubules in Mouse Testis Caused by Active Immunization of Actin-like Protein 7a

作　　者：付俊[1,2] 宗书东 缪时英[1,2] 宋伟[1,2] 

机构地区：[1]中国医学科学院基础医学研究所 [2]北京协和医学院基础学院生物化学与分子生物学系医学分子生物学国家重点实验室,北京100005 [3]国家人口和计划生育委员会科学技术研究所生殖生理学实验室,北京100081

出　　处：《中国医学科学院学报》2013年第6期595-600,共6页Acta Academiae Medicinae Sinicae

基　　金：国家科技支撑计划(2012BAI31B08);国家重点实验室专项基金(2060204)~~

摘　　要：目的原核表达并纯化全长的人精子肌动蛋白样蛋白7a(ACTL7a)蛋白,检测主动免疫ACTL7a产生抗精子抗体后小鼠睾丸的损伤情况。方法构建重组表达质粒pET30a-ACTL7a,以重组质粒转化E.coli Rosseta(DE3),异丙基硫代半乳糖苷(IPTG)诱导目的蛋白表达,通过镍离子金属螯合树脂纯化和SDS-PAGE切胶分离目的蛋白,溶胶后以表达的ACTL7a蛋白免疫ICR小鼠。ELISA检测抗体效价,PAS染色检测主动免疫后曲细精管的发育。结果经IPTG诱导,在大肠杆菌中表达出目的蛋白ACTL7a,纯化后免疫ICR小鼠,经PAS染色发现主动免疫ACTL7a的小鼠其睾丸曲细精管出现生精细胞脱落、核凝聚、生精细胞缺如、生精细胞退化等异常的管腔。结论成功构建了ACTL7a基因全长的原核表达载体,经诱导表达和纯化获得高纯度的目的蛋白。发现ACTL7a蛋白主动免疫ICR小鼠产生抗精子抗体后,小鼠睾丸的曲细精管发生严重损伤。Objective To obtain recombinant sperm-protein actin-like protein 7a （ACTL7a） and de- tect the damage seminiferous tubules in mouse testis caused by anti-sperm antibodies generated by purified ACTL7a active immunization. Methods The recombinant expression plasmid pET30a-ACTL7a was constructed and then transformed into E. coli Rosseta （DE3） . The protein expression was induced by isopropyl 13-D-1-thio- galaetopyranoside （IPTG）, and the protein was purified by nickel ions chelating resin. Finally, the protein was separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis （SDS-PAGE） and harvested by excising the gel containing target. ICR （Institute of Cancer Research） mice were then immunized using purified ACTL7a protein. The antibody titers were determined by ELISA and the development of seminiferous tubules after active immunization was stained by PAS staining. Results Induced by IPTG, the target protein ACTL7a was expressed in E. coll. After purification, it was used to immunize the ICR mice. As shown by PAS staining, spermatid expul- sion, pyknotic cells, absence of germ cells, and germ cells degenerated were seen in the seminiferous tubules in the immunized testes. Conclusions The ACTL7a prokaryotic expression vector was successfully constructed. High- purity target protein was obtained after induction and purification. After the active immunization with the target pro- the seminiferous tubules in the mouse testes will be severely damaged.

关 键 词：肌动蛋白样蛋白7a 表达 纯化 主动免疫 抗精子抗体 

分 类 号：R392.11[医药卫生—免疫学]