[Expression purification and clinical application of high-risk HPV16 E4 gene].
OBJECTIVE
To determine the relationship between serum antibody against HPV16 E4 and cervical cancer, and to construct HPV 16 E4 protein expression vector as an antigen to detect its corresponding serum antibody among different populations.


METHODS
HPV16 E4 early gene was ligated into pRSET-A expression vector. The constructed plasmids were transformed into BL21 (DE3)cells, and induced to express HPV 16 E4 protein by isopropylthio-beta-D-galactoside (IPTG). The expressed E4 inclusions were denatured, purified through Ni-column, and renatured. After the activity was revealed, antibodies against HPV 16 E4 in the sera from healthy women and patients with chronic cervicitis and cervical cancer were respectively determined by enzyme-linked immunosorbent assay (ELISA) using the fusion protein as the antigen.


RESULTS
HPV 16 E4 fusion protein of Mr 15*10(3) was expressed by pRSET-16E4 after IPTG induction. The fusion protein accounted for 30% of the total bacterial proteins and expressed as inclusive body. After purification with Ni-NTA agarose resin, the recombinant protein revealed purity of 95%, and activity of the renatured protein was identified by ELISA. The serum antibody-positive rate of HPV 16 E4 was 10.00%, 39.13% and 28.13%, respectively in 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients. The antibody-positive rate in cervical cancer patients and chronic cervicitis patients were significantly higher than that in healthy women (P<0.01), while the difference between the antibody-positive rate in cervical cancer patients and chronic cervicitis patients was not significant.


CONCLUSIONS
HPV 16 E4 protein expressed from pRSET-A/BL21 can be used in serological studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E4 is present in a significantly higher percentage in cervical cancer and chronic cervicitis patients than in healthy women.
Gao
Yan-e
YE
Department of Obstetrics and Gynecology, Second Affiliated Hospital, School of Medicine, Xioan Jiaotong University, Xioan 710004, China. yanegao@163.com
Hui
Hui
H
Zhang
Ju
J
Fan
Jiang-bo
JB
Yan
Xiao-jun
XJ
D000328:Adult; D000368:Aged; D000914:Antibodies, Viral / Q000097:blood*; D005260:Female; D005822:Genetic Vectors; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D030361:Papillomavirus Infections / Q000276:immunology / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D002583:Uterine Cervical Neoplasms / Q000276:immunology / Q000821:virology*; D002575:Uterine Cervicitis / Q000821:virology
D000914:Antibodies, Viral; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C069119:oncogene protein E4, Human papillomavirus type 16