T载体的构建及日本血吸虫肌动蛋白基因PCR产物的快速克隆  

CONSTRUCTION OF T VECTOR AND RAPID CLONG OF PCR PRODUCTS OF GENE ENCODING SHISTOSOMA JAPONICUM ACTIN

作　　者：沈玉娟[1] 曹建平[1] 刘述先[1] 徐馀信[1] 宋光承[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所

出　　处：《中国血吸虫病防治杂志》2004年第4期277-280,共4页Chinese Journal of Schistosomiasis Control

基　　金：国家"8 63"计划 ( NO.2 0 0 4AA2 15 2 40 );国家自然科学基金 ( NO.3 0 3 712 62 );上海市科委"十五"科技攻关重大计划 ( 0 3 DZ192 3 1)资助~~

摘　　要：目的　构建快速高效克隆 PCR产物的克隆载体 (T载体 ) ,并对日本血吸虫肌动蛋白全长编码基因 PCR产物进行快速克隆。方法　日本血吸虫肌蛋白全长编码基因的扩增采用逆转录 -聚合酶链反应 (RT- PCR)方法。质粒 p GEM5 Zf (+)经限制性内切酶 Eco R V酶切 ,在仅含有脱氧胸苷三磷酸 (d TTP)的 PCR缓冲液中于 70℃作用 2 h,在每个片段的 3′端加上一个脱氧胸苷 (d T)碱基 ,构建成 T载体。根据 PCR扩增产物 3′端存在一个非模板依赖的脱氧腺苷 (d A)原理 ,将扩增产物直接克隆入 T载体并测序。结果　阳性克隆经琼脂糖凝胶电泳、限制性酶切分析、PCR及 DNA序列测定等均证实克隆获得成功 ,且效率很高。与曼氏血吸虫肌动蛋白比较 ,核苷酸和推断的氨基酸的同源性分别是 92 .5 %和 99.7%。结论　构建的 p GEM5 Zf- T载体对日本血吸虫肌动蛋白编码基因的PCR产物直接克隆既经济、简便 ,又快速、高效 ,所构建的 T载体由于在插入位点两侧具有 p UC/M13测序引物序列 ,可直接测定重组质粒中插入片段的核苷酸序列。所获得的日本血吸虫 (大陆株 )Objective To construct pGEM5Zf-T vector and directly clone the polymerase chain reaction products of translate region for Schistosoma japonicum actin into the T vector. Methods The complete gene encoding S.japonicum actin was amplified by RT-PCR. The pGEM5Zf-T vector was prepared by cutting pGEM5Zf(+) vector with EcoR V and adding a 3′ terminal thymidine to both ends by incubating with Taq polymerase in PCR buffer containing 2 mmol/L dTTP for 2 h at 70 ℃. The RT-PCR product was cloned into T vector and sequenced. Results Direct cloning of the PCR products was achieved and identified by agarose gel electrophoresis, digestion with endonucleases, PCR and sequencing. The efficiency of AT cloning was much higher than that of blunt-end ligation. The RT-PCR product, cloned into the T vector, was sequenced and shown to be 92.5% identical at the nuclei acid level and 99.7% identical in deduced amino acid sequence to that of S.mansoni actin. Conclusion The T vector is a convenient system for cloning of PCR products of the gene encoding S.japonicum actin, and the AT cloning is rapid and effective. The recombinant plamids prepared by the AT cloning could be sequenced directly due to the binding site of pUC/M13 sequencing primer within it. The gene encoding actin of S.japonicum is high homologous with that of S.mansoni.

关 键 词：日本血吸虫 肌动蛋白 PCR T载体 测序 

分 类 号：R383.24[医药卫生—医学寄生虫学]