睾丸酮丛毛单胞菌活化因子基因的质粒载体构建及表达  

Construction and Over-expression of the Activator Gene from Comamonas testosteroni

作　　者：戴艺民[1] 潘大仁[1] 吴明霞[1] 周以飞[1] 

机构地区：[1]福建农林大学作物学院

出　　处：《农业生物技术学报》2006年第3期416-422,共7页Journal of Agricultural Biotechnology

基　　金：福建省科技厅攻关计划重大项目(2003N028);福建省发改委项目资助。

摘　　要：提取睾丸酮丛毛单胞菌(Comamonastestosteroni)的基因组DNA,利用PCR扩增activator基因,将扩增产物克隆到pKtac1(含强启动子tac)中,构建重组质粒pKtac1-act1和pKtac1-act2。经酶切分析和测序,鉴定出正确的重组质粒pKtac1-act1和pKtac1-act2。将重组质粒转化入大肠杆菌(Escherichiacoli)HB101中,用酶联免疫分析方法测定细菌总蛋白质中的activator表达量;将pAX1(含3α-HSD/CR基因)分别和重组质粒(pKtac1-act1和pKtac1-act2)一起转化入E.coliHB101中,测定细菌总蛋白质中的3α-HSD/CR和activator的表达量。结果表明:重组质粒能明显提高activator的表达;pAX1与重组质粒共转化的蛋白质粗提物中,activator和3α-HSD/CR的表达量都显著提高。The activator gene from Comamonas testosteroni was amplified by PCR from the genomie DNA of C. testosteroni. The PCR products were cloned into plasmid pKtael （containing tac promoter）, and the recombinant plasmids pKtael-actl and pKtael-act2, were obtained and identified by the analysis of restriction enzymes and DNA sequencing and then respectively transformed into Eschetichia coil HB 101. The total cell lysate of the host bacteria was extracted to detect the quantity of activator using ELISA. Moreover, the recombinant plasmids were also co-transformed with plasmid pAX1 （containing 3α -HSD/CR gene） into E.coli HB 101 respectively. The total cell lysate of the host bacteria was also extracted to detect the quantity of activator and 3α -HSD/CR using ELISA. Moreover, the results indicated that the recombinant plasmids can improve the expression of activator; and the expressions of activator and 3α-HSD/CR can be also improved by plasmid pAXl co-transformed with recombinant plasmids.

关 键 词：睾丸酮丛毛单胞菌 活化因子 3α-羟基类固醇脱氢酶/碳酰基还原酶 基因表达 

分 类 号：S852[农业科学—基础兽医学]