[Steady expression and activity determination of recombinant signal peptide of mBD2 in SiHa cell].
OBJECTIVE
To observe the expression pattern and effect of recombinant murine beta defensin 2 (rmBD2) on the proliferation of cell transfected with pcDNA3. 1 (+)/rmBD2.


METHODS
The recombinant plasmid pcDNA3. 1 (+)/rmBD2 was transferred into SiHa cells. The transfected SiHa cells were selected by G418 with 100 microg/mL for over 20 days. The steady expressions of rmBD2 protein and rmBD2 mRNA were detected by immunofluorescence and RT-PCR. The effects of rmBD2 on SiHa cell growth and reproduction were measured by MTT.


RESULTS
The SiHa cells which could stably express the rmBD2 protein were harvested with 100 microg/mL of G418. The rmBD2 protein expression was, by immunofluorescence, confirmed and its mRNA in SiHa with rmBD2 could be detected at 4 weeks, 6 weeks, 8 weeks, and 10 weeks after cell transfected. A 220 bp segment encoding rmBD2 was amplified by RT-PCR and proved by sequencing. The SiHa/K cells transfected pcDNA3. 1 (+) and SiHa had no expression of rmBD2 protein. The SiHa with rmBD2 expression grew slowly than SiHa/K and SiHa control (P < 0.05).


CONCLUSIONS
The screening for SiHa with rmBD2 expressing stably is successful. The cell growth curve shows that rmBD2 can inhibit the proliferation of tumor cells. The above results establish a solid foundation for further studying the biological properties and the anti-tumor mechanism of beta defensins.
Wei
Xiao-Li
XL
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Li
Ming-Yuan
MY
Li
Hong
H
Shi
Qiao-Fa
QF
Li
Wan-Yi
WY
Jiang
Zhong-Hua
ZH
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D023083:beta-Defensins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
C117047:Defb2 protein, mouse; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D023083:beta-Defensins