[Expression, purification, and serum antibody detection of HPV16 E6 in cervical cancer patients].
OBJECTIVE
Human papillomavirus type 16 (HPV16) is the predominant high-risk type of HPV in cervical cancer tissues. Serum antibody responded to HPV16-related proteins is associated with the development of cervical cancer. This study was to construct and purify recombinant HPV16 E6 protein, detect its corresponding serum antibody among different populations, and explore the correlation of HPV16 E6 serum antibody reaction to cervical cancer.


METHODS
HPV16 E6 early gene was constructed into pRSET-A expression vector. The plasmids were transfected into BL21 (DE3) cells, which were induced to express HPV16 E6 protein by isopropylthio-beta-D-galactoside (IPTG). E6 inclusions were denatured, purified through Ni column, and renatured. After the activity of HPV16 E6 protein being identified, the antibodies against HPV16 E6 in serum samples from 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients were determined by ELISA using the fusion protein as antigen. HPV DNA genotype was estimated in cervical cancer tissues by fluorescence polarization.


RESULTS
HPV16 E6 fusion protein of Mr 24x10(3) was expressed in pRSET-16E6 after induction of IPTG. The fusion protein was accounted for 22.3% of total bacterial proteins, and expressed as inclusive body. After purification with Ni-NTA agarose resin, the purity of the recombinant protein was over 95%, and its activity was identified by ELISA. The antibody-positive rate was significantly higher in cervical cancer patients than in healthy women and chronic cervicitis patients (31.2% vs. 5.0% and 6.5%, P<0.01). In the 32 cervical cancer patients, the positive rates of HPVs DNA and HPV16 DNA in cancer tissues were 90.61% and 46.88%. The antibody-positive rate of HPV16 E6 was higher in HPV16 DNA-positive cervical cancer patients than in HPV16 DNA-negative patients (46.7% vs. 17.6%), but the difference was not significant.


CONCLUSIONS
HPV16 E6 fusion protein obtained from pRSET-A/BL21 can be used in serologic studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E6 is more common in cervical cancer patients than in healthy women and chronic cervicitis patients.
Gao
Yan-E
YE
Department of Obstetrics and Gynecology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi, 710004, P. R. China. yanegao@163.com
Guo
Jin-Zhu
JZ
Zhang
Ju
J
Song
Tian-Bao
TB
Yan
Xiao-Jun
XJ
D000328:Adult; D000368:Aged; D000912:Antibodies, Neoplasm / Q000097:blood*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000821:virology
D000912:Antibodies, Neoplasm; D004279:DNA, Viral; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins