摘要
旨在通过5'-RACE获得美洲大蠊3-磷酸甘油醛脱氢酶(GAPDH)基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,为进一步研究其功能奠定基础。通过3'-RACE技术,PCR扩增获取编码美洲大蠊GAPDH蛋白的全长cDNA序列;采用生物信息学方法推导出该序列编码的氨基酸序列及其理化性质;预测信号肽、蛋白疏水性、可溶性、跨膜区结构、二级结构、三级结构,并构建系统发育树;构建原核表达载体pET28a-GAPDH,IPTG诱导重组蛋白表达,并用His-tag抗体Western blotting验证。结果显示,美洲大蠊GAPDH基因,其完整阅读框含999个碱基,编码332个氨基酸。序列分析显示,该蛋白与家蚕GAPDH相似性为89%,具有GAPDH保守功能域,经IPTG诱导获得重组蛋白。
It was to clone the GAPDH gene of Periplaneta american,analyze its sequence,express recombinant protein of thePeripla-neta americanaGAPDH,predict the protein structure.Base on a fragment sequence of the GAPDH fromPeriplaneta americancDNA library,obtained full-length GAPDH sequence by RACE.The recombinant plasmid pET28a（＋）-GAPDH was built to express His-GAPDH fusion protein in E.coli BL21（DE3）by IPTG induction,and identified by SDS-PAGE and Western blotting.Results showed the Periplaneta american GAPDH gene include an reading fragment of 999 bp code for 332 amino acid residues.It revealed 89% identity with GAPDH gene ofBombyx mori.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第4期113-117,共5页
Biotechnology Bulletin
基金
贵州省社发攻关项目(黔科合S系[2007]1038)
贵阳医学院研究生创新计划专项基金
