家蚕丙酮酸脱氢酶激酶基因的克隆与原核表达  

Cloning and Prokaryotic Expression of Pyruvate Dehydrogenase Kinase Gene in Bombyx mori

作　　者：宋海韬[1] 王力刚[1] 沈兴家[1,2] 黄勇[1] 赵巧玲[1,2] 

机构地区：[1]江苏科技大学,江苏镇江212003 [2]中国农业科学院蚕业研究所农业部蚕桑遗传改良重点开放实验室,江苏镇江212018

出　　处：《江西农业学报》2011年第10期6-10,共5页Acta Agriculturae Jiangxi

基　　金：国家自然科学基金项目(No.NSFC31072083)

摘　　要：丙酮酸脱氢酶激酶(PDK)在糖代谢的调控中具有重要的作用,为了研究家蚕PDK(BmPDK)的功能,以蚁蚕总RNA为模板,应用RT-PCR技术扩增了BmPDK的cDNA,构建原核表达载体pET-28a-BmPDK进行表达和纯化。结果显示,克隆的BmPDK全长cDNA与NCBI公布的序列一致;Western bloting检测表明BmPDK蛋白得到了表达,且主要以包涵体形式存在;IPTG最佳诱导浓度为0.8 mmol/L、最佳诱导时间为8 h;2,6-二氯吲哚酚法间接测定结果表明,纯化的BmPDK蛋白具有酶活性。Pyruvate dehydrogenase kinases（PDK） played an important role in the regulation of sugar metabolism.To study the function of Bombyx mori PDK（BmPDK）,the cDNA of BmPDK was amplified with RT-PCR method by using the total RNA of the newly-hatched silkworm larvae as template,and a prokaryotic expression plasmid pET-28a-BmPDK was constructed for expression and purification.The results showed that the cloned BmPDK cDNA was identical with that published in NCBI.Western bloting indicated BmPDK proteins have been successfully expressed,which existed mainly in the form of inclusion body.The best IPTG concentration and time for inducing the expression of BmPDK was 0.8 mmol/L and 8 hours,respectively.The purified BmPDK showed an enzyme activity when it was examined with the indirect method of 2,6-DCPIP.

关 键 词：家蚕 丙酮酸脱氢酶激酶 原核表达 蛋白质免疫印迹 酶活性 

分 类 号：Q344.13[生物学—遗传学]