蓝氏贾第鞭毛虫alpha-8贾第素特异性锤头状核酶-GCV重组载体的构建  

Construction of GCV-Specific Hammerhead Ribozyme Recombinant Vector of Alpha-8 Giardin in Giardia lamblia

作　　者：魏超君[1,2] 卢思奇[1] 曹利静[3] 田喜凤[4] 

机构地区：[1]甘肃省人民医院临床检验中心,兰州730000 [2]首都医科大学病原生物学系,北京100069 [3]河北省儿童医院重症监护科,石家庄050031 [4]河北联合大学生命科学学院,唐山063000

出　　处：《中国寄生虫学与寄生虫病杂志》2011年第5期321-326,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30970313)~~

摘　　要：目的构建蓝氏贾第鞭毛虫alpha-8贾第素(α-8 giardin)特异性锤头状核酶-GCV重组载体。方法采用RNA draw软件对蓝氏贾第鞭毛虫α-8贾第素基因序列(GenBank登录号为AY781323)的二级结构进行模拟分析,按照G∶C比例和锤头状核酶设计原则,选取合适的核酶切割靶点,设计特异性锤头状核酶(H8)序列,并将其与犬贾第虫病毒(GCV)连接,获得α-8贾第素特异性锤头状核酶-GCV重组载体(pGCV634/H8/1423)。将载体线性化体外转录产物电击转染至贾第虫滋养体细胞内。提取转染后24 h的各组虫体总RNA,并以其为模板采用RT-PCR验证转染效果及对靶mRNA的切割效果。结果成功设计、合成了蓝氏贾第鞭毛虫α-8贾第素mRNA锤头状核酶序列(H8),将其与犬贾第虫病毒载体(GCV)连接,成功构建了pGCV634/H8/1423;RT-PCR实验结果表明,重组载体pGCV634/H8/1423转染贾第虫细胞后24 h可检测到核酶RNA的存在,并实现了对α-8贾第素mRNA高效、特异的切割作用。结论构建的pGCV634/H8/1423能有效转染至贾第虫细胞内,并在其细胞内对α-8贾第素基因的mRNA具有高效、特异的切割作用。Objective To construct a GCV-ribozyme recombinant vectors of α-8 giardin in Giardia lamblia.Methods The secondary structure of α-8 giardin mRNA（GenBank Accession No.AY781323） was analyzed with the RNA draw software.According to the proportion of G ∶ C and principles of designing hammerhead ribozyme,suitable ribozyme cleavage points were chosen.A specific antisense-hammerhead ribozyme（H8）was designed and synthesized.The ribozyme was cloned into Giardia canis virus（GCV） vector to construct a recombinant viral vector-pGCV634/H8/1423.The vector was linearized and transcripted into the trophozoites of G.lamblia by electroporation method.The α-8 giardin mRNA level of the transfectants and normal trophozoites were analyzed 24 h after electroporation by RT-PCR.Results The recombinant vector of GCV-specific hammerhead ribozyme of α-8 giardin in Giardia lamblia（pGCV634/H8/1423） was constructed.RT-PCR assays showed the ribozyme（H8） mRNA can be detected 24 h after transfection and α-8 giardin mRNA was cleaved effectively by ribozyme（H8） introcellularly.Conclusion pGCV634/H8/1423 can transfect Giardia trophozoites and cleave mRNA of α-8 giardin intracellularly.

关 键 词：蓝氏贾第鞭毛虫 贾第素 α-8贾第素 锤头状核酶 犬贾第虫病毒 

分 类 号：R382.213[医药卫生—医学寄生虫学]