[Detection of HPV 58 and cloning of its E7 gene in cervical cancer].
OBJECTIVE
To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer.


METHODS
HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG.


RESULTS
Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins.


CONCLUSIONS
HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.
Gao
Yan-e
YE
Department of Obstetrics and Gynecology, Second Hospital, Xi'an Jiaotong University, Xi'an 710004, China. yanegao@163.com
Zhang
Ju
J
Chen
Zhong-can
ZC
Song
Tian-bao
TB
Zhao
Yan
Y
Zhang
Ge-lin
GL
Yan
Xiao-jun
XJ
D000328:Adult; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005814:Genes, Viral; D006801:Humans; D008875:Middle Aged; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification; D050725:Papillomavirus E7 Proteins / Q000096:biosynthesis* / Q000235:genetics; D030361:Papillomavirus Infections / Q000150:complications / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000821:virology
D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins