睾丸酮丛毛单胞菌teiR基因载体构建及在大肠杆菌中的表达  

Construction of the Plasmid Vector of teiR Gene from Comamonas testosteroni and Its Overexpression in Eschenchia coli

作　　者：陈建秋[1] 潘大仁[2] 周以飞[1] 卢文标[2] 程晓春[2] 王占成[1] 陈雅艳[1] 林钿[1] 

机构地区：[1]福建农林大学作物科学学院,福州350002 [2]福建农林大学生命科学学院,福州350002

出　　处：《农业生物技术学报》2008年第4期681-686,共6页Journal of Agricultural Biotechnology

基　　金：教育部科学技术重点项目(No.207056);福建省科技厅攻关计划重大项目(No.2003N028);福建省教育厅科学技术重点项目(No.JA06011);福建省发改委项目(闽计投资[2003]170号)资助。

摘　　要：提取睾丸酮丛毛单胞菌(Comamonas testosteroni)基因组DNA,利用PCR扩增teiR基因,将扩增产物克隆到pKtac2 (含强启动子tac)和pK18中,构建重组质粒pKtac2-teiR和pKteiR100。将重组质粒分别转化大肠杆菌(Escherichia coli)HB101,提取细菌总蛋白,ELISA测定细菌总蛋白中TeiR蛋白的表达量,结果表明:含tac强启动子的pKtac2-teiR转化大肠杆菌后,TeiR蛋白的表达量可以达到6.65μg/mg,比pKteiR100的表达量高出0.72μg/mg。将p6(含3a-HSD/CR基因)分别和重组质粒(pKtac2-teiR和pKteiR100)共转化大肠杆菌HB101,测定细菌总蛋白中3a-HSD/CR和TeiR的表达量,结果表明:在大肠杆菌中teiR基因可以明显促进3a-HSD/CR基因的表达,pKtac2-teiR与p6共转化大肠杆菌后teiR基因的表达量比pKteiR100与p6共转化后teiR基因的表达量高,其TeiR蛋白表达量分别为5.94和5.33μg/mg;添加终浓度1 mmol/L的IPTG诱导,其TeiR蛋白的表达量分别为6.81和6.10μg/mg。但是pKtac2-teiR与p6共转化后的3a-HSD/CR的表达量低于pKteiR100与p6共转化后的3a-HSD/CR的表达量,未添加IPTG诱导,3a-HSD/CR的蛋白表达量依次为1.20和1.71μg/mg;添加终浓度为1 mmol/L IPTG诱导,其3a-HSD/CR的蛋白表达量依次为1.42和1.80μg/mg。The teiR gene was amplified by PCR from the genomic DNA of Comamonas testosteroni. The PCR products were cloned into plasmid pKtac2（containing tac promoter） and pK18, and the recombinant plasmids pKtac2-teiR and pKteiR100 were constructed and transformed into Escherichia coli HB101, respectively. The total cell lysate of the host bacteria was extracted to detect the expression level of TeiR protein using ELISA. The results showed that the expression level of TeiR protein ofplasmid pKtac2-teiR （containing tac promoter） could reach 6.81μg/mg and it was 0.72μg/mg more than that of the pKteiRlO0 plasmid. Meanwhile, the recombinant plasmids were co-transformed into E. coli HB101 respectively with plasmid p6 （containing 3a-HSD/CR gene）. The total cell lysate of the host bacteria was extracted to detect the expression level of TeiR and 3a-HSD/CR protein using ELISA. The results indicated that teiR gene could significantly promote the 3a-HSD/CR gene expression. The expression level of TeiR protein of plasmid pKtac2-teiR was higher than that of plasmid pKteiR100 aiter co-transformation with plasmid p6. Their expression level could reach 5.94 and 5.33μg/mg, respectively, and was up to 6.81 and 6.10μg/mg aiter inducing with 1mmol/L IPTG. But the expression level of 3a-HSD/CR in E. coli co-transformed with plasmids pKtac2-teiR and p6 was less than that co-transformed with plasmids pKteiR100 and p6. The amounts of 3a-HSD/CR protein were 1.20 and 1.71μg/mg, respectively, and could reach 1.42 and 1.80μg/mg after inducing with 1mmol/L IPTG.

关 键 词：睾丸酮丛毛单胞菌 teiR EuSA 3a-HSD/CR 

分 类 号：S188[农业科学—农业基础科学]