Construction and identification of sense and antisense eukaryotic expression vectors of human vascular endothelial growth factor-C
- VernacularTitle:血管内皮生长因子C正、反义真核表达载体的构建及鉴定
- Author:
Peng ZHU
;
Jianbo ZHANG
;
Yuanbin DAI
- Publication Type:Journal Article
- Keywords:
vascular endothelial growth factor C;
vector construction
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone the vascular endothelial growth factor C gene(VEGF-C),construct its sense and antisense eukaryotic expression vectors,and then to identify and analyze the gene's sequence.Methods Total RNA was isolated from human SGC-7901 cells when the cells were in logarithm growing period.The sense and antisense total length cDNA of VEGF-C gene were then synthesized and amplified from the total RNA by RT-PCR.The plasmid pCI-neo and pcDNA3 were transformed and prepared by Escherichia coli.After being cut with Hind Ⅲ+EcoR I and Xba I+EcoR I respectively,the VEGF-C cDNA was cloned into eukaryotic expression vector pCI-neo and pcDNA3.The recombinant plasmids pcDNA3-sense VEGF-C and pCI-neo-antisense VEGF-C were verified finally by restriction endonuclease analysis and sequencing.Results The positive clone of total length cDNA of VEGF-C gene which had been synthesized and amplified by RT-PCR contained approximately 1.3kb insert.The sense and antisense recombinants were pcDNA3-sense VEGF-C and pCI-neo-antisense VEGF-C.Their positive clone contained an approximately 6.8kb insert.Sequencing of basic radicals confirmed that the sequences of DNA fragments introduced to the recombined plasmids fitted the parameters of the expected design,and the two fragments were complementary with opposite direction.Conclusions The human VEGF-C gene has been cloned and both its sense and antisense eukaryotic expression vectors are constructed successfully,which set up a foundation for the next step of study on the effects of transfected antisense VEGF-C cells on the growth of tumor cells in vitro,and for the further study on the effects of VEGF-C in the formation of lymphatics and metastasis of gastric carcinoma.
