[Prokaryotic expression and identification of human papillomavirus type 16 E5 protein].
OBJECTIVE
To construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH(3)T(3) cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5).


METHODS
HPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plasmid pET32a(+) followed by digestion with BamH I and Hind III. The recombinant plasmid pET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BamH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E. coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH(3)T(3) cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR.


RESULTS
The pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 degrees C, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfected NIH(3)T(3) cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment.


CONCLUSIONS
The prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E .coli and NIH(3)T(3) cells with stable expression of HPV16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.
Shi
Qiao-fa
QF
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. sqf741@sina.com.cn
Wei
Xiao-li
XL
Li
Hong
H
Wang
Bao-ning
BN
Zhang
Wei-dong
WD
Jiang
Zhong-hua
ZH
Cao
Kang
K
Li
Ming-yuan
MY
D016475:3T3 Cells; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D000086082:Viroporin Proteins
D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins