[蓝氏贾第鞭毛虫α-8贾第虫动基体蛋白特异性锤头状核酶重组载体的构建]
[Construction of GCV-specific hammerhead ribozyme recombinant vector of alpha-8 giardin in Giardia lamblia].
作者信息
Wei Chao-Jun, Lu Si-Qi, Cao Li-Jing, Tian Xi-Feng
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2011 Oct;29(5):321-6.
OBJECTIVE
To construct a GCV-ribozyme recombinant vectors of alpha-8 giardin in Giardia lamblia.
METHODS
The secondary structure of alpha-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 transcript into the trophozoites of G. lamblia by electroporation method. The alpha-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 alpha-8 giardin in Giardia lamblia (pGCV634/H8/ 1423) was constructed. RT-PCR assays showed the ribozyme (H8) mRNA can be detected 24h after transfection and alpha-8 giardin mRNA was cleaved effectively by ribozyme (H8) intracellularly.
CONCLUSION
pGCV634/H8/1423 can transfect Giardia trophozoites and cleave mRNA of alpha-8 giardin intracellularly.
目的
构建蓝氏贾第鞭毛虫α-8贾第虫素的GCV-核酶重组载体。
方法
用RNA draw软件分析α-8贾第虫素mRNA（GenBank登录号AY781323）的二级结构。根据G:C比例和锤头状核酶设计原则，选择合适的核酶切割位点。设计并合成特异性反义锤头状核酶（H8）。将核酶克隆到犬贾第虫病毒（GCV）载体中构建重组病毒载体pGCV634/H8/1423。将该载体线性化，通过电穿孔法导入蓝氏贾第鞭毛虫滋养体。电穿孔24小时后，通过RT-PCR分析转染子和正常滋养体中α-8贾第虫素mRNA水平。
结果
构建了蓝氏贾第鞭毛虫α-8贾第虫素的GCV特异性锤头状核酶重组载体（pGCV634/H8/1423）。RT-PCR检测显示，转染24小时后可检测到核酶（H8）mRNA，且核酶（H8）可在细胞内有效切割α-8贾第虫素mRNA。
结论
pGCV634/H8/1423可转染贾第虫滋养体并在细胞内切割α-8贾第虫素mRNA。
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