硫化叶菌病毒dUTPase基因的表达及酶学活性分析  

Expression and enzyme activity of Sulfolobus virus dUTPase gene

作　　者：朱国东[1,2] 魏云林[1] 张琦[1] 季秀玲[1] 林连兵[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500 [2]云南民族大学图书馆,昆明650031

出　　处：《应用与环境生物学报》2014年第4期712-716,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31160035;31260034);中国科学院微生物研究所微生物资源前期开发国家重点实验室开放课题(20100605)资助~~

摘　　要：对硫化叶菌病毒(Sulfolobus virus)STSV2脱氧尿苷焦磷酸酶(dUTPase)基因进行克隆表达,并分析其酶学特征.根据STSV2的dUTPase基因序列设计特异性引物,以病毒STSV2 DNA为模板进行PCR扩增,将产物克隆至原核表达载体pET32a(+)中,然后,将其转化至Escherichia coli BL21(DE3)进行dUTPase的IPTG诱导表达及纯化,通过SDS-PAGE检测其大小,并测定其酶学活性.结果表明,诱导表达的重组dUTPase蛋白分子量(Mr)约为38×103(含pET32a(+)自带的助溶标签),重组dUTPase在Mg2+存在的情况下能特异性催化dUTP生成dUMP和PPi,其反应最适温度为60℃,在pH值3-8之间有较高的活性,其在提高PCR扩增效率和特异性方面具有一定的作用.本研究通过克隆表达获得了硫化叶菌病毒(Sulfolobus virus)STSV2脱氧尿苷焦磷酸酶,其在基因扩增领域有一定的应用价值.This study aimed to understand the expression of dUTPase from Sulfolobus virus STSV2 and to analyze its enzyme characteristics. Primers were designed according to STSV2 dUTPase DNA sequence with dUTPase amplified using STSV2 DNA as template. Target fragment coding dUTPase was cloned into pET32a（＋） plasmid and introduced into E. coli Bl21（DE3）. Expression of dUTPase was induced by Isopropylβ-D-1-thiogalactopyranoside（IPTG） and purified by Ni-NTA spin columns. The molecular weight of dUTPase was analyzed by SDS-PAGE and its activity further detected. The SDS-PAGE result showed that the molecular weight of the recombinant dUTPase was 38 × 10^3. With Mg2＋, the recombinant dUTPase could catalyse dUTP to dUMP and PPi. The optimum enzyme activity of dUTPase occurred at 60 ℃, with high activity kept at pH value from 3 to 8. The recombinant dUTPase also showed specific functions in improving the PCR amplification specificity and specificity efficiency. These results suggested that dUTPase from Sulfolobus virus STSV2 can be obtained by cloning and expression.

关 键 词：硫化叶菌病毒 脱氧尿苷焦磷酸酶 PCR扩增效率 原核表达 酶学活性 

分 类 号：Q786[生物学—分子生物学] Q55