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

发布时间：2019-04-04 12:19

【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。
[Abstract]:Aim to express and purify the full-length human sperm actin-like protein 7a (ACTL7a) protein in E. coli and detect the damage of mouse testis after active immunization with ACTL7a to produce anti-sperm antibody. Methods the recombinant expression plasmid pET30a-ACTL7a, was constructed and transformed into E.coli Rosseta (DE3). Isopropyl galactogalactoside (IPTG) was used to induce the expression of the target protein. The target protein was purified by nickel ion metal chelating resin and the target protein was isolated by SDS-PAGE gel digestion. ICR mice were immunized with ACTL7a protein. Elisa was used to detect antibody titer and PAS staining was used to detect the development of seminiferous tubule after active immunization. Results after induced by IPTG, the target protein ACTL7a, expressed in E. coli was purified and then immunized ICR mice. The results of PAS staining showed that the spermatogenic cells in testicular seminiferous tubule of the active immunized ACTL7a mice were exfoliated, the nuclei condensed and the spermatogenic cells were absent. Abnormal lumen such as spermatogenic cell degeneration. Conclusion the prokaryotic expression vector of the full length of ACTL7a gene was successfully constructed, and the high purity target protein was obtained by inducing expression and purification. It was found that the seminiferous tubules of testis of ICR mice were seriously damaged after ACTL7a protein was actively immunized to produce antisperm antibodies.
【作者单位】：中国医学科学院基础医学研究所;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;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;
【基金】：国家科技支撑计划(2012BAI31B08) 国家重点实验室专项基金(2060204)~~
【分类号】：R363

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