OBJECTIVETo enhance the expression level of human papillomavirus (HPV) 16 L2E7 in Escherichia coli (E. coli), in aim of providing high-level expression of HPV16 L2E7 strain for pre-clinical high-throughout production.

METHODSThe whole L2E7 gene was optimized by software of Synthetic Gene Designer, reflecting E. coli codon usage. Two parts of codon-optimized gene were cloned into pET9a vector step by step. The positive clone, which was sequenced to be corrected, was transfected to BL21 (DE3+) via isopropyl-beta-D-thiogalactoside (IPTG) induction. They produced the HPV16 L2E7 fusion protein, which was further detected by SDS-PAGE and Western blot. The induction temperature, induction time, and IPTG concentration were also optimized by a series of experiments. Further purification modes of this protein were also explored.

RESULTSCodon-optimized HPV16 L2E7 was highly expressed in E. coli. The target protein accounted for nearly 60% of the total cell extract.

CONCLUSIONHigh-level expression of HPV16 L2E7 was successfully constructed.

Codon ; Escherichia coli ; genetics ; metabolism ; Human papillomavirus 16 ; metabolism ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

To investigate E6 and E7 gene variations of human papillomavirus type 16 in Yunnan Province, DNA was extracted from 2000 gynecological outpatient samples. For Human papillomavirus (HPV) genotyping, the genomic DNA was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers, then the PCR products were subjected to direct DNA sequencing. A total of 20 HPV-16 viral DNAs were identified. E6 and E7 genes of HPV-16 viral DNA were then amplified using E6 and E7 specific primers, the PCR products were purified and sequenced. The results showed that mutations were found at nucleotide position 178 of HPV-16 E6 gene in 10 cases,the mutation rate was 50%; For HPV-16 E7 gene, the mutations were found at nucleotide position 647 in 10 cases; the mutation rate was 50%. Phylogenetic analysis showed that Asian (As) variants of HPV-16 were predominated in Yunnan, China. None of African-1, African-2 variants of HPV-16 was found in this region.
Adult ; Base Sequence ; China ; Female ; Genetic Variation ; Human papillomavirus 16 ; classification ; genetics ; isolation & purification ; Humans ; Middle Aged ; Molecular Sequence Data ; Mutation ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; genetics ; Papillomavirus Infections ; virology ; Phylogeny ; Repressor Proteins ; genetics

The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.
Animals ; Cell Line ; virology ; Female ; Human papillomavirus 31 ; genetics ; metabolism ; Humans ; Mice ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus E7 Proteins ; genetics ; metabolism ; Papillomavirus Infections ; virology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

BACKGROUNDHeat shock protein 70 (HSP70) is expressed highly in epithelial tumours associated closely with human papillomavirus 16 (HPV16) infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes.

METHODSStable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic (G418) at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfection was further confirmed by doubly labelled immunofluorescent staining.

RESULTSCompared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfected keratinocytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7.

CONCLUSIONSOur studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.

Cells, Cultured ; HSP72 Heat-Shock Proteins ; biosynthesis ; Humans ; Keratinocytes ; metabolism ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; genetics ; Transfection

OBJECTIVEConducting the gene characterization of the E6 and E7 gene of human papillomavirus 16 (HPV16) isolated from 15 cases of cervical cancer at Beijing.

METHODSOverlapping primers were designed according to the full-length genomes of E6 and E7 from the GenBank and PCR was used to amplify the E6 and E7 fragments. TA clone was used to select a purified clone in order to have better and valuable sequencing results. Nucleotide and amino acid sequence were analyzed by the Sequencer, Bioedit, Mega et al.

RESULTS8 of 15 (8/15) cervical samples contained HPV E6 and E7 gene, and 4 had Asian type like and 4 had Europe prototype like. There were two nucleotide mutation at E6 position 178 (T-->G,D25E) and at E7 position 647 (A-->G, N29S) in 4 Asian type like viruses. There were one nucleotide mutation at E6 position 335 (C-->T, H78Y) in 1 of 4 Europe prototype like virus. In the cervical cancer samples, 8 of 15 contained the HPV16E 6 and E7 gene. HPV16 E6 and E7 can not be detected in denosquamous carcinoma and adenocarcinoma.

CONCLUSIONHPV16 is the main etiology of the cervical carcinoma. The HPV16 infectious ratio of squamous carcinoma is more than the ratio of adenocarcinoma. 178th nucleotide in E6 gene is the very important site to distinguish the Asia and the Europe prototype strain like. 178 nucleotide in E6 and 647 nucleotide in E7 are the frequent mutation site in cervical carcinoma. Analysis based on the E6 and E7 gene sequence of HPV 16 isolates suggests that naturally occurring sequence variants of E6 and E7 gene may have identify the oncogenic properties.

Adenocarcinoma ; virology ; Carcinoma, Squamous Cell ; virology ; China ; Female ; Human papillomavirus 16 ; chemistry ; genetics ; isolation & purification ; Humans ; Mutation ; Oncogene Proteins, Viral ; chemistry ; genetics ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; virology ; Repressor Proteins ; chemistry ; genetics ; Sequence Homology, Amino Acid ; Uterine Cervical Neoplasms ; virology

OBJECTIVETo investigate the correlation between HPV16 and the development of laryngeal squamous cell carcinoma by studying the effects of HPV16 E6 and E7 oncogene on the proliferation and differentiation of human laryngeal squamous carcinoma cell line.

METHODSCationic phosphonolipid was used to transfect pLXSN16E6E7 into Lscc-02 high differentiation laryngeal squamous cell carcinoma cell line, the cells transfected by the vector pLXSN and Lscc-02 cells served as control groups. The changes of proliferation and differentiation were measured in vitro and in vivo.

RESULTSThe proliferation was quicker in experimental group. The average A ratio reached to 2.96, which was higher than 1.90 and 1.85 of control groups. The clone forming rate was 23.6% in experimental group, which was higher than 12.7% and 12.0% of control groups. Serum-dependent became lower in experimental group. The Ki-67 positive expression rate was markedly increased and cytokeratin 13 positive expression rate was markedly decreased in experimental group as compared with control groups. The Ki-67 positive expression rates were 93.8%, 80.7% and 79.2% respectively. The cytokeratin 13 positive expression rate were 80.9%, 91.0% and 93.7% respectively. The latent period and internal double time of transplant tumor were short in experimental group compared with control groups. The latent period were 19 d, 28 d and 30 d respectively. The internal double time of transplant tumor was 2.15 d, 3.28 d and 3.47 d respectively. The volume of transplant tumor was smaller in the same period in experimental group. The cells of transplant tumor in experimental group showed an image of small size, great allotype and a trend of low differentiation.

CONCLUSIONSHPV16 E6 and E7 can promote the proliferation and inhibit the differentiation of Lscc-02 laryngeal squamous cell carcinoma cells which suggest HPV16 may play an important role in the development of laryngeal squamous cell carcinoma.

Carcinoma, Squamous Cell ; genetics ; virology ; Cell Differentiation ; genetics ; Cell Line, Tumor ; Human papillomavirus 16 ; genetics ; Humans ; Laryngeal Neoplasms ; genetics ; virology ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; genetics ; Repressor Proteins ; genetics

BACKGROUNDTo select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer.

METHODSE6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity.

RESULTSWestern blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.

Animals ; Antibodies, Viral ; biosynthesis ; Female ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Insertional ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; Vaccinia virus ; immunology ; Zinc Fingers

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.
Cancer Vaccines ; biosynthesis ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Female ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Papillomavirus Vaccines ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Uterine Cervical Neoplasms ; prevention & control ; Vaccines, DNA ; biosynthesis

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.
Animals ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Female ; Mice ; Mice, Inbred C57BL ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo establish an immortalized oral epithelial cell line.

METHODSNormal human oral epithelial cells were transfected with HPV16E6/E7 open reading frames using recombinant retroviral system pLXSN. Expression of HPV16E6 and E7 protein were tested by Western blot in three kinds of cells. To define cellular biological characterization of HPV16E6/E7 transfected cells, a series analysis were performed, including protraction of growth curve, HE staining, immunocytochemical staining and scanning electron microscope observation. The tumorigenicity was assessed by colony formation and transplanting the cells into nude mice.

RESULTSHuman oral epithelial cells transfected with HPVE6/E7 has been in culture for over 18 months. The cell line was named HIO615. Western blot analysis showed HIO615 expressed HPV16 E6 and E7 protein. HIOC were positive for cytokeratin, tonofibril and desmosome as observed by scanning electron microscope. The number of large colonies of dense multilayer cells was low (0.77%). No tumor developed in nude mice injected subcutaneously with HIOEC.

CONCLUSIONA human immortalized oral epithelial cell line induced by HPV16E6 and E7 has been successfully established.

Animals ; Cell Line, Transformed ; Cell Transformation, Viral ; Humans ; Mice ; Mice, Inbred BALB C ; Mouth Mucosa ; cytology ; ultrastructure ; virology ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins