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

OBJECTIVE: Persistent infection of HPV increases the chance of carcinoma in situ of cervix through stages of cervical intraepithelial neoplasia (CIN) 1, 2, and 3, and finally progresses into cervical cancer. We aimed to explore the safety and efficacy of BLS-M07 which is orally administered agent expressing human papillomavirus (HPV) 16 E7 antigen on the surface of Lactobacillus casei in patients with CIN 3. METHODS: Patients with CIN 3 were recruited in our clinical trial. Reid Colposcopic Index (RCI) grading and serum HPV16 E7 specific antibody production were used to evaluate efficacy of BLS-M07. In phase 1, BLS-M07 was administered orally, 5 times a week, on weeks 1, 2, 4, and 8 with dosages of 500 mg, 1,000 mg, and 1,500 mg. In phase 2a, patients were treated with 1,000 mg. The primary endpoints were the safety and the pathologic regression on colposcopic biopsy. RESULTS: Nineteen patients were enrolled in the CIN 3 cohort. In phase 1, no patients experienced dose limiting toxicity. No grade 3 or 4 treatment-related adverse events or deaths were observed. At 16 weeks after treatment, RCI grading was improved and serum HPV16 E7 specific antibody production increased (p<0.05). Six of 8 (75%) patients with CIN 3 were cured in phase 2a. CONCLUSIONS: Oral immunization with BLS-M07 increases production of serum HPV16 E7 specific antibody which induces protective humoral immunity. The safety of this oral vaccine was proved and could be a competitive non-surgical therapeutic agent of CIN 3. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02195089
Antibody Formation ; Biopsy ; Carcinoma in Situ ; Cervical Intraepithelial Neoplasia ; Cervix Uteri ; Cohort Studies ; Female ; Humans ; Immunity, Humoral ; Immunization ; Lactobacillus casei ; Papillomavirus E7 Proteins ; Papillomavirus Vaccines ; Uterine Cervical Neoplasms

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.
Animals ; Capsid Proteins ; biosynthesis ; Codon ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Human papillomavirus 16 ; Mice ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; biosynthesis ; Papillomavirus E7 Proteins ; biosynthesis ; Papillomavirus Vaccines ; therapeutic use ; Recombinant Fusion Proteins ; biosynthesis

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

OBJECTIVETo explore the in vitro inhibitive effect and underlying mechanisms of Brucea Javanica oil emulsion (BJOE) on human papilloma virus (HPV) type 16 infected cells.

METHODSThe HPV16 E61E7 immortalized human ectocervical Ect1/E6E7 cell line and the CaSki cell line were selected as the in vitro models of premalignant cervical lesion and cervical cancer respectively. After treated with BJOE at different concentrations (5, 10, 20, and 40 microg/mL) at the operation time points (24, 48, and 72 h), the effects of BJOE on proliferative activities were measured by MTT assay. The morphologic changes of cell apoptosis stained with Hochest 33,258 were observed by fluorescence microscope. The effect on the cell apoptosis rate was analyzed by Annexin V-FITC/PI double-labeled flow cytometry. The mRNA expressions of HPV16 E6 and E7 were determined by semi-quantitative RT-PCR. The protein expressions of HPV16 E6, E7 oncogene, and specifically interacted p53, Rb antioncogene were stained by immunocytochemical staining (Elivison two-step procedure).

RESULTS(1) The proliferative activities of the Ect1/E6E7 cell and the CaSki cell treated with BJOE at different concentrations (5, 10, 20, and 40 p g/mL) at the operation time points (24, 48, and 72 h) were obviously inhibited, showing dose- and time-dependent manners (P <0.05). (2) Typical changes of apoptosis were observed in both HPV 16 positive cell lines after treated with BJOE. The cell apoptosis rates increased markedly after being cultured with BJOE at different concentrations (5, 10, and 20 microg/mL) for 48 h (P < 0.05). (3) After treated with BJOE at different concentrations (5, 10, and 20 microg/mL) for 48 h, the HPV16 E6 and E7 mRNA relative expressions in both HPV 16 positive cell lines decreased significantly (P < 0.05). (4) After treated with BJOE at different concentrations (5, 10, and 20 microg/ mL), the expressions of HPV16 E6, E7, and mutant p53 protein decreased gradually (P < 0.05), while the Rb protein expression increased gradually (P < 0.05).

CONCLUSIONSBJOE showed obvious in vitro inhibitory effects on HPV type 16 infected cells. Its underlying mechanisms might be possibly associated with down-regulating expressions of HPV16 E6 and E7 oncogenes.

Apoptosis ; drug effects ; Brucea ; chemistry ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Female ; Human papillomavirus 16 ; drug effects ; pathogenicity ; Humans ; Oncogene Proteins, Viral ; metabolism ; Papillomavirus E7 Proteins ; metabolism ; Papillomavirus Infections ; Plant Oils ; pharmacology ; Repressor Proteins ; metabolism

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

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

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

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