[Establishment of Hela cell line inducibly expressing human active granzyme B].
OBJECTIVE
To construct inducible expression vector for human granzyme B gene and express it in Hela cell line.


METHODS
Human active granzyme B gene was obtained by PCR and cloned into the inducible expression vector pIND. The resulting expression vector, together with a helper plasmid pVgRXR, was stably transfected into Hela cells using Lipofectamine 2000. The transfected cells were selected in medium containing G418 and zeocin. The resistant cells were induced with ponasterone A, and the optimal concentration of ponasterone A and time of induction were determined by immunocytochemical staining. Then the effects of the expressed granzyme protein on Hela cells were detected by MTT colorimetry and cytoskeletal staining.


RESULTS
The Hela cells that inducibly expressed human active granzyme B were obtained. Induction with 30 micromol/L ponasterone A for 5 days caused the strongest expression of granzyme B. The induced cells appeared as either multinucleate giant cells or pyknotic small cells, and their growth was inhibited. Further analysis demonstrated cytoskeletal abnormality of multinucleate giant cells.


CONCLUSIONS
The establishment of inducible expression system for active granzyme B lays the foundation for further research on biological function of granzyme B.
Wang
Zhi
Z
Department of Biochemistry and Molecular Biology, Faculty of Preclinical Medicine, Fourth Military Medical University, Xi'an 710032, China.
Zhao
Jing
J
Zhang
Li
L
Yu
Cui-juan
CJ
Wang
Cheng-ji
CJ
Yang
An-gang
AG
D049109:Cell Proliferation; D003599:Cytoskeleton; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics; D053804:Granzymes; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D014162:Transfection
D004441:Ecdysterone; C008999:ponasterone A; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases