人Izumo4和Tssk6 cDNA的重组及其多克隆抗体的制备

Human Izumo4 and Tssk6 cDNA recombinant and the preparation of its polyclonal antibody

目的研究Izumo4和Tssk6在精卵融合中的相互作用。方法根据世界卫生组织标准选取的正常精液标本取自内蒙古医科大学附属医院生殖医学中心。SPF级昆明种小鼠5只,雄性,4~6周龄,体质量18~22 g。利用精液提取RNA,克隆人类Izumo4和Tssk6的c DNA序列,原核表达,通过重组质粒p MD-Izumo4、重组质粒p MD-Tssk6转染BL21（DE3）感受态菌制备HIS-Izumo4和HIS-Tssk6;然后通过小鼠腹腔制备小鼠抗人Izumo4和Tssk6腹水多克隆抗体。结果提取的人精子RNA在260 nm光密度值与280 nm光密度值比值为1.86、1.87,提取物无蛋白和杂质。克隆出正确的Izumo4和Tssk6 c DNA序列的p MD-Izumo4和p MD-Tssk6重组质粒。p ET-Izumo4和p ET-Tssk6重组载体在E.coli BL21（DE3）中复制,表达HIS-Izumo4和HIS-Tssk6融合蛋白,其相对分子质量分别位于35 000~45 000、45 000~55 000。2次切胶后,获得较纯的HIS-Izumo4/Tssk6融合蛋白。通过小鼠腹腔免疫,获得可以识别人内源性Izumo4和Tssk6蛋白的小鼠抗人HIS-Izumo4/Tssk6腹水多克隆抗体。结论实验制备小鼠抗人HIS-Izumo4/Tssk6腹水多克隆抗体,可特异性识别人的Izumo4/Tssk6蛋白,可以用于对人HIS-Izumo4/Tssk6蛋白的研究。

Objective To study the interaction of Izumo4 and Tssk6 in sperm-oocyte fusion. Methods Normal semen sam- ples were obtained from Reproductive Medicine Center of Inner Mongolia Medical University Affiliated Hospital according to World Health Organization standard. Five SPF Kunming male mice, 4 - 6 weeks old and with body weight of 18 - 22 g were collected. RNA was extracted from semen, and human Izumo4 and Tssk6 cDNA sequence was cloned and prokaryotic ex- pressed. Mouse HIS-Izumo4 and HIS-Tssk6 were prepared by recombinant plasmid pMD-Izumo4, pMD-Tssk6 recombinant plasmid transfected BL21（DE3） competent bacteria. The Izumo4 and Tssk6 aseites polyclonal antibody were prepared in mouse peritoneal. Results The ratio of optical density at 260 nm and 280 nm in RNA extracted from human sperm without protein and impurities in the extract was 1.86 and 1.87. Correct pMD-Izumo4 and pMD-Tssk6 plasmid clone of Izumo4 and Tssk6 cD- NA sequences was prepared. The pET-Izumo4 and recombinant vector pET-Tssk6 Exoli BL21（DE3） expressed HIS-Izumo4 and HIS-Tssk6 fusion protein, the molecular weights were between 35 000 - 45 000, 45 000 - 55 000, which were consistent with expectations. The size-matched pure HIS- Izumo4/Tssk6 fusion protein was obtained after twice gel slices. HIS-Izumo4/Tssk6 ascites polyclonal antibody of human Izumo4 and Tssk6 protein were prepared. Conclusion It is demonstrated that mouse anti-human HIS-Izumo4/Tssk6 ascites polyclonal antibody have strong recognition specificity in human Izumo4/Tssk6 pro- tein, and can be used for the study of HIS-Izumo4/Tssk6 protein.