[Construction and expression of recombinant antibody/granzyme B containing truncated translocating peptide].
OBJECTIVE
To construct an eukaryotic expression vector for recombinant antibody/granzyme B gene containing truncated translocating peptide and express it in HER-2(+) SKBR-3 cells and HER-2(-) Hela cells.


METHODS
PCR amplication was used to obtain recombinant DNA encoding antibody/granzyme B containing truncated translocating peptide DNA, and then the DNA fragment was cloned into eukaryotic expression vector pCMV-e23sFv-PE40. After being transfected into SKBR-3 cells and Hela cells, the expression of target gene and its effect on cellular morphology were detected by immunocytochemical staining.


RESULTS
The eukaryotic expression vector encoding recombinant antibody/granzyme B containing truncated translocating peptide was successfully constructed. e23sFv-FSD-GrB protein was expressed in most of Hela cells and had no effect on Hela cells, but the transfected SKBR-3 cells which also express e23sFv-FSD-GrB protein exhibited condensed nucleus and cytoplasm.


CONCLUSIONS
The construction of eukaryotic expression vector of recombinant antibody/granzyme B gene containing truncated translocating peptide lays the foundation for further determination of minimal fragment responsible for translocation.
Zhang
Li
L
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China. agyang@fmmu.edu.cn
Zhao
Jing
J
Wang
Zhi
Z
Wen
Wei-hong
WH
Zhang
Ying-hua
YH
Wang
Cheng-ji
CJ
Yang
An-gang
AG
D036002:ADP Ribose Transferases / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D005098:Exotoxins / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D053804:Granzymes; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D037521:Virulence Factors / Q000235:genetics*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D053804:Granzymes; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C509213:GZMB protein, human