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KMID : 0391319940040020241
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Korean Journal of Biological Response Modifiers
1994 Volume.4 No. 2 p.241 ~ p.250
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Biochemical characterization of recombinant HPV18 E7 protein and its use for the diagnosis of HPV18 infection
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강주현
박종섭/유왕돈/김승호/윤희식/함경수/박순희
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Abstract
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Background:
@EN Human Papillomavirus (HPV) type 16 and 18 are strongly associated with cervical carcinoma and cervical intraepithelial neoplasia. It is necessary to develop the diagnostic tools for detection of HPV infections and prophylatic vaccines and
therapeutic vaccines. HPV18 E7 protein binds pRB105 and the ability to bind pRB105 is essential for the transforming and immortalizing activities of E7. In addition, E7 is capable of transactivating the adenovirus E2 promote. The two Cys-X-X-Cys
motifs
in the C-terminal half of E7 are important for this activity. We confirmed that the purified recombinant HPV18 E7 protein can be associated with pRB105 (retinoblastoma susceptability gene product) and have metal binding property. To study the
relationship of incidence of cervical carcinoma and infection of human papillomavirus, we serodiagnosed the cervical cancer patients using the purified E7 protein as antigen.
@ES Methods:
@EN To serodiagnose, the purified E7 protein was electrotransferred to nitrocellulose filter and reacted with 100-fold diluted human sera as primary antibodies. We confirmed that the purified HPV18 E7 protein can be associated with pRB105 of HeLa
cell
line by immunocoprecipitation method using protein A Sepharose and monoclonal antibody to pRB105. To observe the metal binding property of E7 protein, ultraviolet absorbance spectroscopy was performed.
@ES Results:
@EN Isoelectric point of purified E7 protein was approximately 4.7. Sera for 4 cases among 38 cervical cancer patients were reacted with the purified E7 protein in immunostaining analysis but sera for 10 healthy women were not. This result
suggests
that
10.5% of cancer patients may be infected with HPV18. The recombinant purified HPV18 E7 protein can be associated with pRB105 of HeLa cell by immunocoprecipitaion method using protein A-Sepharose and monoclonal antibody to pRB105. The spectra
obtained
with purified E7 protein incubated with 1 to 30 molar equivalents of zinc ion showed that the absorbance at near 250mnm as zinc was added.
@ES Conclusion:
@EN It was important to show that sera from cervical cancer patients would react with the HPV open reading frame (ORF) recombinant protein expressed in E. coli. Immunostaining analysis using the purified E7 as antigen suggested that 10.5% of
cervical
cancer patients (38 cases tested) was infected with HPV18. Highly purified recombinant E7 protein that are shown be able to bind pRB105 suggesting that this protein might maintain functional conformation. Absorbance enhancement of Zn-E7 mixture
at
near
250nm in the spectra indicated that HPV18 E7 protein purified in E. coli is cysteine-rich zinc-binding protein.
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KEYWORD
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