[Construction of GCV-specific hammerhead ribozyme recombinant vector of pyruvate kinase in Giardia lamblia]

To construct a recombinant vector of Giardia canis virus (GCV)-specific hammerhead ribozyme of pyruvate kinase (PK) in Giardia lamblia.

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.

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%.

The recombinant vector can transfect Giardia trophozoites, and cleave mRNA of PK intracellularly and extracellularly.