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.

Fig 1. Construction of the expression vector of the HPV58 E7.

(A) Construction of pGEX-4T2-(HPV58-E7) vector. The lane M1 shows the TaKaRa DL2000 DNA marker, lane 1 shows the HPV58 E7 gene (PCR from human HPV58-positive cervical epithelial cells); lane 2 is the pGEX-4T2 vector; lane 3 is the cleaved pGEX-4T2 vector by EcoR I and BamH I; lane 4 shows the reconstructed pGEX-4T2-(HPV58-E7) vector; lane 5 is the verification of pGEX-4T2-(HPV58-E7) vector which was cleaved by EcoR I and BamH I; the lane M2 is TaKaRa DL 15,000 DNA marker. (B) Construction of pEGFP-C1-(HPV58-E7) vector. The lane M1 shows the TaKaRa DL2000 DNA marker, lane 1 shows the HPV58 E7 gene (PCR from pGEX-4T2-(HPV-58E7) vector); lane 2 is the pEGFP-C1 vector; lane 3 is the cleaved pEGFP-C1 vector by EcoR I and BamH I; lane 4 shows the reconstructed pEGFP-C1-(HPV58-E7) vector; lane 5 is the verification of pEGFP-C1-(HPV58-E7) vector which was cleaved by EcoR I and BamH I; the lane M2 is TaKaRa DL 15,000 DNA marker.

Fig 2. Expression and purification of HPV58 E7 protein.

(A) Coomassie blue staining for recombinant GST-HPV58-E7 protein. Lane M is the protein ladder; Lane 2 shows the protein of the supernatant of the bacterial lysate after centrifugation; Lane 3 shows the protein of the sediment of bacteria lysate. The arrowhead indicates GST-HPV58-E7 protein. (B) Coomassie blue staining for purified HPV58-E7 protein. Lane M is the protein ladder; Lane 1 and 2 show the purified E7 protein after removal of GST-tag; Lane 3 and 4 show the E7 protein after dialysis in PBS. The arrowhead indicates HPV58-E7 protein.

Fig 3. Verification of polyclonal antibody against HPV58 E7.

(A) western blotting for the detection of HPV58 E7. Lanes 1, 2 and 3 are the cell lysates of HEK293T, 293T-pEGFP-C1, and 293T-pEGFP-C1-HPV58-E7, respectively; Lane 4 is the purified HPV58-E7 protein. (B) Immunofluorescent analysis for the detection of HPV58 E7 in 293T-pEGFP-C1, 293T-pEGFP-C1-HPV58-E7 cells. (C) Immunohistochemistry staining for the detection of HPV58 E7 in cervical cancer cells. Red arrowheads indicate the HPV58-positive cells. White arrowheads indicate the HPV58-negative cells.

Fig 4. Examination of the cross-reaction of HPV 58 E7 antibody.

(A) western blotting for the detection of HPV58 E7. Lanes 1, 2 and 3 are the pure protein of HPV 58, 16 and 18 E7. (B) Immunofluorescent analysis for the detection of HPV58 E7 in SiHa and HeLa cells. (C) Immunohistochemistry stain for the detection of HPV58 E7 in cervical cancer cells, which was HPV 16- or 18-positive, HPV 58 was negative. 1 is the HPV16-positive sample and 2 is the HPV18-positive sample.