家兔Ⅰ型肽载体(PepTⅠ)基因的克隆与原核表达

Cloning and prokaryotic expression of rabbit peptide transporter 1

用real-time PCR方法扩增家兔PepTⅠmRNA序列,通过原核表达体外获得家兔PepTⅠ蛋白,旨在制备多克隆抗体。试验通过RT-PCR方法成功扩增出2 001bp的家兔PepTI基因片段,设计特异性引物成功扩增出抗原表位相对集中的1 071bp大小的片段,构建重组表达质粒,获得融合蛋白。结果显示:家兔PepTⅠ基因片段成功的连接到pMD18-T载体上,重组克隆载体双酶切后回收目的片段分别连接到原核表达载体pET-32a、pET-28a,经转化提取质粒测序验证后表明,成功构建原核表达载体pET-32a-P、pET-28a-P,将重组质粒转化至感受态细胞BL21(DE3)后IPTG诱导,成功表达出PepTⅠ融合蛋白,目的蛋白相对分子质量大小约为44 000,与预期试验结果相符。结果表明:本试验成功扩增出家兔PepTⅠmRNA序列,并且通过原核表达获得家兔PepTI融合蛋白。

This study aims to gain the PepTⅠ mRNA sequence and the PepT1 protein of rabbit with real time PCR and primary applicationin vitro to prepare the polyclonal antibody.This study amplified a 2001 bp sequence of rabbit peptide transporter 1（PepT1）gene successfully with RTPCR.A 1 071 bp fragment was amplified successfully with specific primers that antigenic determinant concentrated relatively and was used to construct the recombinant expression plasmid and obtain the fusion protein.The part of PepT1 gene was cloned into the pMD18-T successfully.The recombination carrier was digested with BamHⅠand XholⅠrestriction enzyme and the target fragment was gathered and connected to pET-32 aand pET-28 arespectively to get prokaryotic expression vectors.After the transformation,extraction and sequencing of the above plasmids,it was showed that the prokaryotic expression vectors of pET-32a-P and pET-28a-P were constracted successfully.The recombinant plasmids were transformed to BL21（DE3）,which were competent cells,and induced by IPTG.In BL21（DE3）,the PepT1 fusion protein was expressed successfully,whose molecular weight was 44 000 and the result was accorded with what we expected.In this study,the PepT1 mRNA of rabbit was amplified successfully and the rabbit PepT1 fusion protein was gain by prokaryotic expression.