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[Construction of GCV-specific hammerhead ribozyme recombinant vector of alpha-8 giardin in Giardia lamblia]
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.
