Construction of eukaryotic expression vector of hMTH1 gene antisense RNA.
- Author:
Gao-feng JIANG
1
;
Zhi-xiong ZHUANG
;
Qi-zhan LIU
;
Yun HE
;
Liu-tao DU
Author Information
- Publication Type:Journal Article
- MeSH: DNA Repair Enzymes; Genetic Vectors; genetics; Humans; Phosphoric Monoester Hydrolases; genetics; Plasmids; RNA, Antisense; biosynthesis; Reverse Transcriptase Polymerase Chain Reaction
- From: Chinese Journal of Industrial Hygiene and Occupational Diseases 2003;21(1):57-60
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA.
METHODSThe conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection.
RESULTS423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%.
CONCLUSIONThe efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.
