广州管圆线虫GAMMA-BBH基因的克隆表达鉴定和荧光定量检测  

Identification and FQ-PCR nvestigation of GAMMA-BBH Gene of Angiostrongylus cantonensis

作　　者：孟锦绣[1] 何蔼[2] 程梅[2] 甘明[2] 徐贵峰[2] 李卓雅[2] 余细勇[1] 蒋文玲[1] 李运雄[1] 詹希美[2] 

机构地区：[1]广东省人民医院医学研究中心,广州510080 [2]中山大学基础医学院寄生虫学教研室,广州510080

出　　处：《热带医学杂志》2007年第7期613-617,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金-广东省政府联合基金(No.U0632003);国家"十五"科技攻关重大项目(No.2003BA712A03-07);广州市科技计划项目(No.2005Z3-C7561;No.2006Z3-C7191);广东省医学科研基金(No.B2006003);广东省人民医院科学技术研究基金(No.Y200532)。

摘　　要：目的亚克隆和表达广州管圆线虫γ-丁基甜菜碱羟化酶(γ-butyrobetaine hydroxylase,GAMMA-BBH)基因,对该蛋白属性进行鉴定,定量检测不同虫龄该目的基因表达量。方法PCR扩增GAMMA-BBH cDNA基因,产物经NcoⅠ和HindⅢ限制性内切酶酶切后连接至原核表达载体重组为pET-BBH,在BL21(DE3)中用IPTG诱导表达,SDS-PAGE、Western blot和酶活性测定鉴定表达产物,用荧光定量PCR方法检测广州管圆线虫不同虫龄GAMMA-BBH的表达量。结果PCR和核苷酸序列测定结果表明pET-BBH构建正确并能在IPTG诱导下表达,SDS-PAGE和Western blot鉴定表明表达产物分子量与预期分子量(Mr=48500)一致,酶学测定显示表达产物有GAMMA-BBH的酶活性,荧光定量PCR方法检测结果显示GAMMA-BBH在虫体不同虫龄期表达量不一致。结论目的基因能正确编码并表达具有酶活性的GAMMA-BBH,该酶的表达量与虫龄和寄生部位可能有关。Objective To study the target gene of A ngiostrongylus cantonensis on γ-butyrobetaine hydroxylase （GAMMA-BBH）property by subcloning and expressing and to quantify its expression in different worm stage. Methods The target cDNA was amplified by PCR and PCR products were indigested by restriction enzyme Nco Ⅰ and Hind Ⅲ. Then the indigested products were recombined into pET-BBH and pET-BBH was expressed in E. coli BL21 （DE3） by IPTG inducing. Next, the expressed proteins were identified by SDS-PAGE, western blot and enzyme activity test. Finally, fluorescence quantitative PCR was used to test its expression quantity in different worm stages. Results PCR and nucleotide sequencing results Showed that the aim gene was correctly recombined to be pET-BBH and it could be expressed in vitro. The protein molecular weight was in accord with the GAMMA-BBH molecular weight 48.5 kDa. Enzyme activity test showed that the expression product has GAMMA-BBH enzyme activity. FQ-PCR results suggested there are distinguish expressing levels in different worm periods. Conclusion The aim gene codes GAMMA-BBH protein of A. cantonensis and its expression level may relate to worm ages and parasite condition.

关 键 词：广州管圆线虫 γ-丁基甜菜碱羟化酶 克隆 表达 

分 类 号：R735[医药卫生—肿瘤]