人细胞核自身抗原精子蛋白的重组表达及多克隆抗体制备

目的:获得纯化的人细胞核自身抗原精子蛋白(hNASP)及其多克隆抗体,为其功能研究做准备。方法:提取人睾丸组织总RNA,用自行设计的引物,PCR扩增hNASP的一段序列,PCR产物经TA克隆后,通过BamHⅠ和HindⅢ双酶切克隆到pET-28 a(+)中。在E.coliBL21中,用异丙基-β-硫代半乳糖苷(IPTG)诱导表达H is融合蛋白。样品超声处理后,经镍离子亲和树脂进行亲和层析纯化。用纯化的重组蛋白免疫家兔获取多克隆抗体。结果:对表达重组蛋白的质粒进行DNA测序以及表达的重组蛋白经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,证实获取了目的蛋白。ELISA证实免疫家兔后获得高效价的抗体。结论:用上述原核生物表达的方法可以得到纯化的hNASP蛋白,用纯化的蛋白免疫家兔也能获得高效价的抗体。

Cloning,Expression and Purification of Human Nuclear Autoantigenic Sperm Protein(hNASP) and Preparation of Its Polyclonal Antibody

Objective: To acquire the purified human nuclear autoantigenic sperm protein(hNASP) and its polyclonal antibody for investigating the possible functions of hNASP involved in fertilization.Methods: The coding sequence of hNASP gene was amplified from human testis RNA with specific primers,and the PCR product was cloned first into pMD-18T and then into pET-28a(+) after restriction digestion with BamHⅠand Hind Ⅲ.The fusion protein was expressed in E.coli BL21(DE3) after induction with IPTG.The recombinant protein NASP was purified from the supernatant with Ni~(2+)-NTA His-bind resin under native conditions.Results: The results of DNA sequencing and SDS-PAGE analysis showed the protein to be what we had hoped to acquire.ELISA showed that we had acquired rabbit anti-hNASP serum with high titer.Conclusion: High purity recombinant hNASP protein could be obtained with the above-mentioned prokaryotic expression method,and so could the rabbit anti-hNASP serum with high titer and high specificity.