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


CONCLUSIONS
The recombinant vector can transfect Giardia trophozoites, and cleave mRNA of PK intracellularly and extracellularly.
Cao
Li-jing
LJ
Pediatric Intensive Care Unit, Hebei Children's Hospital, Shjiiazhuang 050031, China.
Feng
Xian-min
XM
Wei
Chao-jun
CJ
Wang
Feng-yun
FY
Zang
Xi-chen
XC
Lu
Si-qi
SQ
D005822:Genetic Vectors; D016829:Giardia lamblia / Q000201:enzymology / Q000235:genetics; D019198:Giardiavirus / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D011770:Pyruvate Kinase / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D049452:Green Fluorescent Proteins; D011770:Pyruvate Kinase