[Expression of a chimeric gene containing poly-arginine as the protein transduction domain and its killing activity against HER2 positive gastric cancer SGC-7901 cells].
OBJECTIVE
To construct a eukaryotic expression vector for chimeric gene containing poly-arginine as the protein transduction domain(PTD) and transiently transfect this vector into HER2 positive SGC-7901 cells and HER2 negative HeLa cells to examine its effect on cell growth.


METHODS
PCR amplication was used to obtain the gene of active form caspase-3 fused with nonaarginine, and then fusion gene was cloned into eukaryotic expression vector containing e23sFv DNA fragment. After this chimeric gene transfected into SGC-7901 cells and HeLa cells by Lipofectamine 2000™; reagent, indirect immunofluorescence and cell counting were used to examine the expression in these two cells and the effect on cell growth.


RESULTS
The eukaryotic expression vector, named pCMV-e23sFv-R₉;-casp3, encoding e23sFv/caspase-3 containing nonaarginine as the PTD was successfully constructed. e23sFv- R₉;-casp3 protein was expressed in a secretary manner in both SGC-7901 cells and HeLa cells. Transfected SGC-7901 cells were found obvious growth inhibitory, morphology change and condensed nucleus, whereas neither growth inhibitory nor apparent morphology change was detected in transfected HeLa cells.


CONCLUSIONS
Of the secretary expressed chimeric protein, the antibody moiety against HER2 can mediate targeted recognition, the nonaarginine translocating peptide can promote activation and translocation of the effector molecule, and the active caspase-3 can effectively induce cell killing.
Zhao
Zhi-ning
ZN
Department of Immunology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Wang
Tao
T
Ren
Jun-lin
JL
Zhang
Rui
R
Zhao
Jing
J
Wen
Wei-hong
WH
Jia
Lin-tao
LT
Yang
An-gang
AG
D017209:Apoptosis; D053148:Caspase 3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology*
D053148:Caspase 3; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C015462:polyarginine; C508053:ERBB2 protein, human