蓝氏贾第鞭毛虫丙酮酸激酶特异性锤头状核酶-GCV重组载体的构建  

Construction of GCV-Specific Hammerhead Ribozyme Recombinant Vector of Pyruvate Kinase in Giardia lamblia

作　　者：曹利静[1,2] 冯宪敏[3] 魏超君[2] 王凤云[2] 张西臣[4] 卢思奇[2] 

机构地区：[1]河北省儿童医院重症监护科,石家庄050031 [2]首都医科大学寄生虫学教研室,北京100069 [3]吉林医药学院病原学教研室,吉林132013 [4]吉林大学动物科学与畜牧兽医学院,长春130062

出　　处：《中国寄生虫学与寄生虫病杂志》2010年第2期98-102,138,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30670224)~~

摘　　要：目的构建蓝氏贾第鞭毛虫丙酮酸激酶(pyruvate kinase,PK)特异性锤头状核酶-犬贾第虫病毒(Giardia canis virus,GCV)重组载体。方法采用RNAdraw软件分析贾第虫编码丙酮酸激酶的基因序列,并设计特异性反义锤头状核酶(PKH)序列,将其与犬贾第虫病毒(GCV)连接,构建重组载体pGCV-PKH。将重组载体线性化体外转录产物,分别进行贾第虫细胞外、细胞内目的mRNA切割实验,采用荧光显微镜观察转染后24h的各组虫体。采用实时PCR对切割产物进行相对定量分析。结果构建了载有蓝氏贾第鞭毛虫丙酮酸激酶特异性锤头状核酶的犬贾第虫病毒重组载体pGCV-PKH。细胞内切割实验结果表明,荧光显微镜下只有pGCV-GFP转染组虫体显示绿色荧光,pGCV-PKH转染组丙酮酸激酶mRNA的相对含量约为正常对照组的33.14%。细胞外切割实验结果表明,该组载体能在细胞外有效切割丙酮酸激酶mRNA,在设定条件下,其切割效率为58.5%。结论重组载体pGCV-PKH能有效转染蓝氏贾第鞭毛虫细胞,并能在其细胞内外对丙酮酸激酶mRNA进行有效切割。Objective To construct a recombinant vector of Giardia canis virus （GCV）-specific hammerhead ribozyme of pyruvate kinase （PK） in Giardia lamblia. Methods Using RNA draw software, the secondary structure of PK gene in Giardia was predicted and the cleavage site of ribozyme was selected. The antisense-specific hammerhead ribozyme （PKH） targeting this site was designed. The DNA encoding the ribozyme was synthesized and the recombinant vector pGCV-PKH was constructed. The extracellular and intracellular cleavage of PK mRNA was carried out with the linear recombinant vector. Trophozoites in each group were observed with fluorescent microscopy at 24th hour after transfection. Real-time PCR was used for relative quantitative analysis of cleavage products. Results The recombinant vector of GCV-specific hammerhead ribozyme of pyruvate kinase in Giardia lamblia （pGCV-PKH） was constructed. Intracellular cleavage assays showed that the green fluorescence could only be seen in pGCV-GFP transfected trophozoites. The relative content of PK mRNA in pGCV-PKH transfected group was 33.14% of that in normal control group. It could cleave PK mRNA extracellularly and the efficiency was 58.5%. Conclusion The recombinant vector can transfect Giardia trophozoites, and cleave mRNA of PK intracellularly and extracellularly.

关 键 词：蓝氏贾第鞭毛虫 犬贾第虫病毒 锤头状核酶 丙酮酸激酶 

分 类 号：R382.213[医药卫生—医学寄生虫学]