Production of Polyclonal Antibody to the HPV58 E7 Protein and Its Detection in Cervical Cancer

Abstract

The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer worldwide, and HPV type 58 is the third most common HPV type in eastern Asia. The E7 oncoprotein is constitutively expressed in HPV58-associated cervical cancer cells and plays a key role during tumorigenesis. To study the biological function of HPV58 E7 and to characterize E7 protein-host cell interactions, we cloned the human HPV58 E7 gene and produced specific E7 antibodies. The HPV58 E7 gene was cloned into a prokaryotic expression vector, pGEX-4T2. The recombinant plasmid pGEX-4T2-(HPV58-E7) was transformed into Escherichia coli DH5α and expressed as a fusion protein containing a GST tag. After purification and removal of the GST affinity tag, the E7 protein was used as an antigen for the production of antiserum in rabbits. The specificity of the purified HPV58 E7 antibody was detected by western blotting, immunofluorescence and immunohistochemistry analysis. These methods demonstrated that the polyclonal antibody could specifically recognize the endogenous and the recombinant HPV58 E7 proteins. Immunohistochemistry analysis indicated that the E7 protein was localized in the nucleus of cervical cancer cells.

Construction of a Recombinant OmpC Dominant Epitope-Based Vaccine Against Escherichia coli and Evaluation of Its Immunogenicity and Protective Immunity

“…The process for preparation of monoclonal antibodies is complex (28), and polyclonal antibodies have the advantages of low cost, shorter time period, and high effect (29). Mouse polyclonal antibodies are commonly used in immunological studies (30). In this study, we obtained a specific antiserum using the method of mouse immunization.…”

Immunodiagnosis and Immunotherapeutics Based on Human Papillomavirus for HPV-Induced Cancers

“…In this method, an IgG2a-type anti-HPV16 E7 mAb was used as the detection antibody, and an IgM-type anti-HPV16 E7 mAb was used as the capture antibody ( 21 ). A Rabbit polyclonal antibody to the HPV58 E7 protein was also developed for detection of cervical cancer; it can specifically recognize the endogenous and the recombinant HPV58 E7 proteins ( 33 ).…”

HPV58 E7 Protein Expression Profile in Cervical Cancer and CIN with Immunohistochemistry

“…FFPE HPV58-positive (detected by PCR) cervical cancer or CIN sections (8-µm) were used for immunohistochemical staining of the HPV58-E7 protein 9 , anti-p16 mouse monoclonal antibody (mAb) (1:500, GT201302, Gene, Shanghai, China) and anti-Ki-67 mouse mAb (1:500, MAB-0672, MXB, Fuzhou, China). Endogenous peroxidases in the de-paraffinized sections were quenched with 0.3% hydrogen peroxide in 60% methanol for 20 min.…”

“…To date, a commercial antibody to HPV58 E7 for immunohistochemistry detection is still unavailable. We have produced and verified the specific polyclonal antibody against the HPV58 E7 protein 9 . In this study, we aimed to detect the HPV58 E7 protein expression using this antibody in the tissues of HPV58-positive cervical cancer and CIN with IHC.…”