小鼠PRL-3原核表达载体的构建  

The Construction of the Prokaryotic Expression Vector for Mouse PRL-3

作　　者：曹燕飞[1] 吕娟 寿成超 

机构地区：[1]长治医学院生物化学教研室,046000 [2]北京大学临床肿瘤学院

出　　处：《长治医学院学报》2012年第6期404-406,共3页Journal of Changzhi Medical College

摘　　要：目的:构建小鼠PRL-3基因的原核表达载体,并在感受态细菌BL21中表达。方法:提取小鼠黑色素瘤B16F10细胞的总RNA,经RT-PCR扩增小鼠PRL-3基因,并将其克隆入原核表达载体pGEX-4T-1中,阳性克隆经限制性内切酶BamHI、EcoRI酶解及测序鉴定后,转化感受态细菌BL21,经IPTG诱导表达GST-mPRL-3融合蛋白。结果:获得全长为522bp的小鼠PRL-3基因片段,构建重组质粒pGEX-4T-1-mPRL-3,转化感受态细菌BL21后,经IPTG诱导,表达出分子质量约为46KD的GST-mPRL-3蛋白。SDS-PAGE分析显示,表达的蛋白主要以可溶性形式存在于E.coli BL21的胞质中。结论:成功地构建了原核表达载体pGEX-4T-1-mPRL-3,为抗体的制备筛选和肿瘤的相关研究奠定了实验基础。Objective： To construct the prokaryotic expression vector for mouse PRL-3, express recombinant GST- mPRL 3 in compenent cells E. coli BL21. Methods：The coding region was amplified from total RNA extracted from mouse melanoma ceils B16F10. The mPRL 3 gene had been cloned into pGEX-4T-1 expressing vector. The positive clone was analyzed by Ban：HI and EcoRI digestion and DNA sequencing. The fusion protein was expressed in BL21 strain and identified by SDS-PAGE. Results：The prokaryotic expression vector was constructed and confirmed with BamHI and EcoRI digestion and by DNA sequencing. The expression vector was transformed into E. coli BL21 and fusion protein GST mPRL 3 was mainly expressed in cytoplasm of BL21 in the soluble form after induction with IPTG. The relative molecular weight of the protein was about 46 kd. Conclusion：The prokaryotic expression vector for mPRL-3 had been constructed successfully and laid a foundation for the research of antibody preparation and screening and tumor.

关 键 词：小鼠PRL-3 原核表达载体 融合蛋白 

分 类 号：R349.52[医药卫生—基础医学]