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  

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

作　　者：王旻[1] 石建莉[1] 程国艳[1] 胡燕琴[1] 刘春萌[1] 徐晨[1] 

机构地区：[1]上海交通大学医学院组织学与胚胎学教研室,上海市生殖医学重点实验室,上海200025

出　　处：《中华男科学杂志》2006年第10期867-871,共5页National Journal of Andrology

基　　金：国家自然科学基金(30470909);上海市"登山行动计划"基础研究重点项目(06JC14046)

摘　　要：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。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 BamH I and 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 Ni2＋ -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.

关 键 词：人细胞核自身抗原精子蛋白 基因克隆 蛋白表达 多克隆抗体 

分 类 号：Q819[生物学—生物工程]