αB-晶状体蛋白CRYAB基因重组表达载体的构建及分子生物学鉴定

Construction and Identification of the Expression Recombinant of αB-Crystallin CRYAB Gene

目的:构建人晶状体蛋白CRYAB基因与真核表达载体pIRES2-DsRed-Express的重组体,为研究人晶状体蛋白CRYAB基因的功能奠定基础.方法:根据人晶状体蛋白CRYAB基因的核苷酸序列,设计并合成分别带有酶切位点的引物,从pMD 18T-CRYAB基因克隆载体中扩增出CRYAB基因外显子片段,与载体pIRES2-DsRed-Express连接构建人晶状体蛋白CRYAB基因的表达载体,转化入大肠杆菌DH5α中.经抗生素筛选阳性克隆,通过酶切图谱分析、菌落PCR和测序鉴定所构建的表达载体.结果:构建的载体经PCR、酶切鉴定和测序证实插入方向正确,表达阅读框正确,载体构建成功.结论:重组人晶状体蛋白CRYAB基因表达载体的构建为研究CRYAB基因的功能及进一步研究先天性白内障的发病机制奠定了基础.

This study aims to construct the recombinant of CRYAB gene, which was inserted into pIRES2-DsRed- Express vector, for further study of CRYAB gene and its function. The procedure consists of the following steps ： （1） Designing and synthesizing primers with specific restriction enzyme sites based on the sequence of human crystallin CRYAB gene; （2） Amplifying the target gene fragment from plasmid pMD 18T-CRYAB, （3） Inserting the PCR products to the digested pIRES2-DsRed-Express vector; （4） Analyzing the positive colony and identifying the recombinants by restriction enzymes ,conlony PCR and DNA sequensing. The results show that the expression vector of CRYAB gene was constructed successfully. It is concluded that expression recombinant of αB-crystallin CRYAB gene will be helpful in studying function of CRYAB gene.