title: [Prokaryotic expression and identification of HPV16 E7 protein].
abstract: HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine.
authors.affiliation: Jiangsu Key Laboratory of Zoonosis; Ministry of Education, Key Lab for Avian Preventive Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Jia
authors.forename: Yue-Lan
authors.initials: YL
authors.lastname: Yin
authors.forename: Chun-Guang
authors.initials: CG
authors.lastname: Bai
authors.forename: Hong
authors.initials: H
authors.lastname: Fu
authors.forename: Yun-Fei
authors.initials: YF
authors.lastname: Gao
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Pan
authors.forename: Xin-An
authors.initials: XA
authors.lastname: Jiao
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D050725:Papillomavirus E7 Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins; C059731:oncogene protein E7, Human papillomavirus type 16