Cyr61蛋白表达载体的构建及表达

Vector Construction and Expression of Cysteine Rich Protein 61(Cyr61) cDNA

目的:体外表达并纯化Cyr61蛋白片段。方法:提取乳腺癌组织总RNA,用一步法RT-PCR选择扩增编码Cyr61蛋白的cDNA片段,并将之克隆到融合蛋白表达载体pQE80L中,转染大肠杆菌Rosetta-gamiTM2。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:克隆到编码Cyr61蛋白片段的cDNA片段,其大小为930bp。构建的表达质粒pQE80-Cyr61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32kD的可溶性融合蛋白,经蛋白质印迹分析鉴定为Cyr61的融合蛋白。结论:本实验克隆了编码Cyr61蛋白片段的cDNA序列,并成功地获得了可溶性表达的融合目的蛋白,为进一步制备抗Cyr61蛋白的抗体和建立Cyr61蛋白定量检测方法创造了条件。

Objective.To express and purify Cyr61 in vitro. Methods.Total RNA was extracted in breast cancer tissue. The gene sequences which code Cyr61 fragment were amplified with one- step RT- PCR. The cDNA were inserted into prokaryotic expression vector pQE80L, then transformed into competent E. coil Rosetta- gami TM 2 cells. 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： 930 bp cDNA of Cyr61 was obtained. In pQE80L expression system, fusion proteins of Cyr61 was obtained, with a molecular weight of 32 kD. Conclusion：The expression vectors of Cyr61 was constructed and the fusion proteins of it was obtained, which sets up basis of producing anti - Cyr61 monoclonal antibody and quantifying of Cyr61 protein.