title: Human papillomavirus type 16 E6, E7 and L1 and type 18 E7 proteins produced by recombinant baculoviruses.
abstract: Proteins derived from the E6, E7 and L1 ORFs of HPV16 and the E7 ORF of HPV18 were produced in insect cells using a baculovirus expression system. HPV ORFs were inserted into baculovirus transfer vectors pAcYM1 or pVL1393/2, and recombinant baculoviruses isolated using a combination of limiting dilution and plaque assay. Using HPV-specific antisera and monoclonal antibodies HPV proteins were identified in lysates of Spodoptera frugiperda (Sf-21) cells infected with HPV-recombinant baculovirus. Immunoreactive HPV16 E7 protein produced in Sf-21 cells had an apparent M(r) of 19 kDa, larger than that predicted from the amino acid sequence, and similar to that of native HPV16 E7 protein in HeLa and CaSki cells. The apparent M(r) of recombinant HPV18-E7, HPV16-L1 and HPV16-E6 proteins was equivalent to the M(r) values predicted from the amino acid sequence. Thermostability studies revealed that the half-life of HPV16-E7 protein in Sf-21 cell lysate was approx. 20 h at 4 degrees C, 2 h at 22 degrees C, and less than 30 min at 37 degrees C. HPV16 L1, HPV16 E7 and HPV18 E7 proteins were predominantly localised in the nucleus of recombinant baculovirus-infected Sf-21 cells, whereas recombinant HPV 16 E6 protein was localised in both the cytoplasm and nucleus of infected insect cells. Northern blot analysis of RNA derived from insect cells infected with vAc16E6E7, a recombinant baculovirus containing both HPV16 E6 and E7 ORF's, revealed the presence of only E6 ORF transcripts, suggesting that the splicing of RNA products derived from the E6 and E7 ORF's, as observed in cervical cancer-derived cell lines, is not performed in insect cells. Baculovirus-derived HPV proteins have similar biological properties to the native proteins and should be suitable for studies on the immunology of HPV.
authors.affiliation: Papillomavirus Research Unit, Lions Human Immunology Laboratories, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Australia.
authors.forename: D S
authors.initials: DS
authors.lastname: Park
authors.forename: L A
authors.initials: LA
authors.lastname: Selvey
authors.forename: S R
authors.initials: SR
authors.lastname: Kelsall
authors.forename: I H
authors.initials: IH
authors.lastname: Frazer
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D036022:Capsid Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D009856:Oncogene Proteins, Viral / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins; D014162:Transfection
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; C063317:L1 protein, Human papillomavirus type 16