多囊蛋白1表达载体的构建及表达

Vector construction and expression of polycystin-1 extracellular region cDNA

目的 :体外表达并纯化多囊蛋白 1胞外区片段。方法 :提取健康人肾组织总 RNA,用一步法 RT- PCR选择扩增编码多囊蛋白 1胞外多拷贝区的 2个 c DNA片段 ,并将之克隆到融合蛋白表达载体 p QE30中 ,转染含有阻遏质粒 p REP4的大肠杆菌 M15。异丙基硫代半乳糖苷 (IPTG)诱导表达融合蛋白 ,用亲和层析法纯化。纯化产物经聚丙烯酰胺凝胶电泳及蛋白质印迹分析鉴定。 结果 :克隆到 2个编码多囊蛋白 1胞外区的 c DNA片段 ,其大小分别为 5 0 2 bp和 4 71bp。构建的表达质粒p QE30 - PK D1e1和 p QE30 - PK D1e2经限制性内切酶酶切和 DNA测序证实为所需要的质粒。表达出相对分子质量分别为19 80 0、1890 0的融合蛋白 ,经蛋白质印迹分析鉴定为多囊蛋白 1的融合蛋白。结论 :本实验克服了 PK D1基因在体内多个同源序列的干扰 ,克隆了编码多囊蛋白 1胞外多拷贝区的 c DNA序列 ,并成功地获得了融合表达的目的蛋白 ,为进一步制备抗多囊蛋白

Objective:To express and purify polycystin 1 extracellular region in vitro .Methods:Total RNA was extracted in kidney tissue of a healthy man.Two gene sequences which code polycystin 1 extracellular region were amplified with one step RT PCR.The fragments were inserted into prokaryotic expression vector pQE30,then transformed into competent E.coli M15 cells which contained repressor(pREP4) plasmid.The fusion protein expression was induced by IPTG and purified by affinity chromatography.The products were identified by SDS PAGE and Western blot analysis.Results:502 bp and 471 bp cDNA of polycystin 1 extracellular region were obtained.In pQE30/pREP4 expression system,fusion proteins of polycystin 1 extracellular region were obtained,with a molecular weight of 19.8 kD and 18.9 kD,respectively.Conclusion: The expression vectors of polycystin 1 extracellular region is constructed and the fusion proteins of it is obtained, this sets up basis of producing anti polycystin 1 monoclonal antibody.