蓝氏贾第鞭毛虫PPDK特异性锤头状核酶-GCV重组载体的构建及鉴定  

CONSTRUCTION AND CHARACTERIZATION OF GCV RECOMBINANT VECTOR-MEDIATED HAMMERHEAD RIBOZYME FOR GIARDIA LAMBLIA PPDK

作　　者：曹利静[1] 冯宪敏[1] 卢思奇[1] 张西臣[2] 王凤云[1] 

机构地区：[1]首都医科大学病原生物学系,北京100069 [2]吉林大学畜牧兽医学院,长春130062

出　　处：《寄生虫与医学昆虫学报》2008年第1期1-7,共7页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金资助项目(No.30670224)

摘　　要：丙酮酸磷酸双激酶(Pyruvatephos phate dikinase,PPDK)可能是蓝氏贾第鞭毛虫能量代谢中具有催化作用的关键酶桩一。为了进一步探讨该酶在贾第虫能量代谢中的作用,本文采用RNA draw软件分析贾第虫编码PPDK的基因序列并设计特异性反义锤头状核酶(Hammerheade ribozyme),克隆该核酶序列并与犬贾第虫病毒(GCV)连接,构建了载有特异性锤头状核酶的贾第虫病毒重组载体pGCV634/H5/2174。该载体经线性化处理后进行体外转录,转录产物以电击方式转染对数生长期的贾第虫滋养体。提取转染后24h虫体总RNA,并以其为模板进行RT-PCR验证转染效果和对靶mRNA的切割效果。结果初步证实了该载体对虫体细胞内编码PPDK的mRNA具有切割作用。Previous extracellular experiments indicated that pyruvate phosphate dikinase （PPDK） has catalytic effect and estimated that it plays an important role in the energy metabolism of Giardia lamblia, which has not been demonstrated by the intracellular evidence yet. In order to understand its function in the energy metabolism of this organism, we analyzed the sequence encoding Giardia PPDK with the software RNA draw, designed and synthesized a specific antisense-hammerhead ribozyme （designated H5）. The ribozyme was cloned into Giardia canis virus vector to constructe a recombinant viral vector-pGCV634/H5/2174. The vector was linearized and transcripted, then introduced into the log-phase growing tropbozoites of G. lamblia by electroporation method. The PPDK mRNA level of the transfectants and normal trophozoites were anlalyzied 24 h after electroporation by RT-PCR. The results showed that the PPDK mRNA level of the transfectants was decreased remarkably or eliminated, compared with the normal trophozoites. This indicated that the mRNA of PPDK was cleavaged by the hammerhead ribozyme （HS） mediated by G.canis virus.

关 键 词：蓝氏贾第鞭毛虫 丙酮酸磷酸双激酶 锤头状核酶 贾第虫病毒 

分 类 号：R382[医药卫生—医学寄生虫学]