淮南麻鸭IGF-Ⅰ基因的克隆、序列分析及原核表达

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of IGF-ⅠGene in Huainan Duck

本研究旨在分析淮南麻鸭IGF-Ⅰ基因的结构特点以及获得编码的蛋白。根据GenBank数据库中鹅、鸡和鸭的IGF-Ⅰ基因序列,设计1对引物P1/P2,利用RT-PCR方法克隆了淮南麻鸭的包含有编码区的部分IGF-Ⅰ基因。将IGF-Ⅰ编码区基因连接到原核表达载体pPROEXTMHTb,然后转化到感受态菌DH5α中,经终浓度为1.0 mmol/L的IPTG诱导表达IGF-Ⅰ融合蛋白。结果表明:获得了534 bp的淮南麻鸭IGF-Ⅰ部分基因,经过生物信息进行序列分析,包括1个462 bp的开放阅读框,可编码153个氨基酸,与鸭、鸡、人、绵羊、牛、野猪和大鼠的同源性分别达到99%、99%、84%、81%、83%、84%和78%;经SDS蛋白质电泳结果表明,成功获得分子量约27 ku的IGF-Ⅰ融合蛋白,纯化后经Western-blotting鉴定,可与兔抗人IGF-Ⅰ抗体发生特异性免疫反应。

The research aimed to study the structural characteristics of IGF- I gene in Huainan duck and the functional production and analysis of its encoding proteins. According to the published sequence of IGF- I in GenBank, a pair of primers P1/P2 was designed to clone and analyze cDNA sequence of IGF-I gene in Huainan duck using RT-PCR and bioinformaties technique. The recombinant expression vector was constructed by cloning IGF- I gene in Huainan duck into the prokaryotie expression plasmid pPROEXTMHTb. The resulted showed that a eDNA sequence with the size of 534 bp was obtained, which included a 462 bp whole ORF and encoded 153 amino acid residuals, has an overall similarity with comparable region of domestic duck （99%）, chicken （99%）, coturnix （99%）, human（84%）, sheep（81%）, bovine（83%）, pig （84%） and rat（78%）. The recombinant vector was transformed into the E. coli DH5 （ strain. The fusion protein is 27ku in size and it can affect specific reaction with the antibody through Western-blotting.