溶脲脲原体蛋白UreG抗体对小鼠体外受精的影响

目的:探索与人精子膜存在交叉反应的溶脲脲原体蛋白UreG抗体对小鼠体外受精的影响。方法:提取溶脲脲原体总DNA,用自行设计的引物扩增UreG全序列,PCR产物经TA克隆后,用BamHⅠ和HindⅢ双酶切,克隆到表达载体pET-28a(+)中。在E.coliBL21中,异丙基-β-硫代半乳糖苷(IPTG)诱导下表达His融合蛋白。用亲和层析纯化的重组蛋白免疫BALB/c小鼠,获取多克隆抗体。观察该抗体对小鼠体外受精的影响。结果:对表达重组蛋白的质粒进行DNA测序以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,证实获取了目的蛋白。ELISA检测证实,免疫小鼠产生了高效价的抗体。该抗体能够抑制小鼠精卵的融合。结论:与人精子膜存在交叉反应的溶脲脲原体蛋白UreG抗体,可导致小鼠精卵结合率降低。

Effect of Antiserum against UreG on Mouse in vitro Fertilization

Objective: To explore the effect of UreG, which is a cross-reactive antigen between Ureaplasma urealyticum(U.U) and human sperm membrane, on mouse in vitro fertilization. Methods: The coding sequence of UreG gene was amplified from U.U with specific primers. The PCR product was first cloned into pMD-18T and then cloned into pET-28a (+) after restriction digestion with BamHI and Hind III. The fusion protein was expressed in E. coli BL21 (DE3) after induction with IPTG. The recombinant protein was purified with affinity chromatography. The mouse polyclone antiserum was produced with purified recombinant UreG as antigen. The IVF was performed to observe the effect of the antibody against recombinant UreG on mouse fertility. Results: The results of DNA sequencing and SDS-PAGE analysis showed that the protein was what we had expected to acquire. ELISA showed that mouse anti- UreG serum with high titer was raised. The antiserum inhibited mouse sperm-egg fusion. Conclusion: The antiserum against UreG decline the mouse sperm-egg fertilization rate.