This study was intended to establish a method of purification of HPV16 L1 protein expressed in a prokaryotic system and to obtain the purified protein. The prokaryotic expression vector pGEX-4T-1-HPV16 L1 was constructed and transformed into E. coli BL21 cell, and induced by 1 mM IPTG to express HPV16L1 protein. The inclusion bodies were isolated and solubilized with 8 M urea. After the urea was removed by gradual dialysis, the denatured L1 protein were renatured and then were purified by affinity chromatography. The results showed that HPV16L1 protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify HPV16L1 protein and hence provide a basis for studying the applications of HPV16 L1 protein.
Capsid Proteins ; biosynthesis ; Escherichia coli ; metabolism ; Genetic Vectors ; Human papillomavirus 16 ; Oncogene Proteins, Viral ; biosynthesis

OBJECTIVETo express the L1 protein of human papillomavirus type 16 (HPV16) in insect cell suspension culture system.

METHODSOptimized the conditions of suspension culture, recombinant virus amplification and protein expression. Determined the virus tilter by plague analysis and detected the target protein by SDS-PAGE and Western blot; The formation of VLPs by HPV16 L1 protein was observed with TEM.

RESULTSThe Sf9 cells could grow better in suspension culture with seeding density of 5 x 10-5 cell/mL and the maximum expression quantity was obtained by infection of cells with rBacV/HPV16L1 (MOI =10) and harvesting after 72-84 h. HPV16L1 protein could assemble into VLPs in Sf9 cells observed with TEM.

CONCLUSIONThe conditions of cell culture, virus amplification and protein expression were optimized. HPV16 L1 protein could assemble into VLPs in Sf9 cells, which would provide a foundation for further study of the vaccine and diagnosis kits.

Animals ; Capsid Proteins ; biosynthesis ; Cell Proliferation ; Oncogene Proteins, Viral ; biosynthesis ; Recombinant Proteins ; biosynthesis ; Spodoptera ; Suspensions ; Virion ; ultrastructure

OBJECTIVETo study the potential transcriptional depression activities of HPV2 E2 proteins with mutations in different functional domains.

METHODSThe primers for constructing various E2 mutants were synthesized based on a HPV2 isolate containing several point mutations within E2 open reading frame. Different E2 mutations were generated by the method of extending PCR and inserted into plasmid pcDNA3. 1. Various recombinant mammalian expression plasmids pcDNA3. 1-E2 were co-transfected into HeLa cells together with a CAT-reporter plasmid pBLCAT-LCR containing HPV-2 prototype LCR, respectively. The transcriptional repression activities of the E2 mutants were evaluated by detection of CAT expression values.

RESULTSCompared with the full-length prototype E2, removals of both N- and C-terminal domains abolished E2 transcriptional repressive activities. The point mutations in the transactivation domain (nt 3037), the internal hinge region (nt 3387) and DNA binding domain (nt 3697) showed remarkable inhibition on its transcriptional depression function.

CONCLUSIONThe transcriptional regulation activity of HPV2 E2 is related with its DNA binding and transactivation domains. The exchanges of the single amino acid within E2, derived from a HPV2 isolate, abolish significantly the repressive effect on viral promoter in the context of full-length E2.

HeLa Cells ; Humans ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; genetics

To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(=6.397,=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(=8.241,=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(=2.916,=0.0434;=3.452,=0.0260). In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.
DNA Damage ; DNA Replication ; Human papillomavirus 16 ; Mitosis ; Oncogene Proteins, Viral

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

OBJECTIVE: To investigate the molecular mechanism of Wnt5a and Epstein-Barr virus latent membrane protein 1 (LMP1) aberrant expression in the nasopharyngeal carcinogenesis and to estimate if it can act as a molecular marker for nasopharyngeal cancer (NPC). METHODS: Immunohistochemistry combined with previously made tissue microarrays were used to study the expression of Wnt5a and LMP1 in the nasopharyngeal carcinogenesis tissues. We investigated the role of over expression of Wnt5a and LMP1 in the development and progression of NPC and their relation with the clinicopathological features of NPC and whether they could act as molecular markers in benign and malignant NPC. RESULTS: The positive percentage of Wnt5a and LMP1 protein expression in the NPC was significantly increased as compared with that in atypically hyperplastic nasopharyngeal epithelium, hyperplastic nasopharyngeal epithelium and histologically normal nasopharyngeal epithelium (P<0.05, P<0.01, and P<0.01). Wnt5a and LMP1 proteins were significantly higher in atypically hyperplastic nasopharyngeal epithelium than those in the hyperplastic nasopharyngeal epithelium and normal nasopharyngeal epithelium (P<0.05 and P<0.01). The positive expression of Wnt5a and LMP1 proteins in clinical T3 and T4 staged NPC was higher than that in clinical T1 and T2 staged NPC (P<0.01 and P<0.05). The positive expression of Wnt5a protein in the NPC with lymph node metastasis was higher than that in the NPC without lymph node metastasis (P<0.01). The positive percentage of LMP1 protein was significantly increased in non-keratinizing carcinoma compared with undifferentiated carcinoma and keratinizing carcinoma (P<0.05 and P<0.05). The expression of Wnt5a protein in the NPC had significant positive correlation with LMP1 (r=0.354, P<0.001). Combined molecular phenotype of both Wnt5a and LMP1 expression was a good marker to distinguish NPC from non-cancerous nasopharyngeal epithelium. CONCLUSION The expression of Wnt5a and LMP1 protein in the NPC is positively correlated, and both wnt5a and LMP1 protein play important roles in the nasopharyngeal carcinogenesis either together or successively promoting the malignant transformation of nasopharyngeal epithelium and the development and progression of NPC. Both Wnt5a and LMP1 positive expression may act as good markers for NPC differential diagnosis.
Biomarkers, Tumor ; genetics ; metabolism ; Carcinogenesis ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Oncogene Proteins, Viral ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Tissue Array Analysis ; Viral Matrix Proteins ; genetics ; metabolism ; Wnt Proteins ; genetics ; metabolism ; Wnt-5a Protein

OBJECTIVETo research the application of the single time detection of HPV E6/E7 mRNA and HC2-HPV-DNA in cervical screening project.

METHODSWe detected both HPV E6/E7 mRNA and HC2-HPV-DNA of each sample which collected from 130 cervical disease patients' cervix during Jan. 2008 and July. 2009. TCT results were taken as standard to evaluate the diagnostic accuracy of the above two test methods in detecting high-grade cervical disease.

RESULTS82.3% (107/130)women were confirmed to infect HPV by HC2-HPV-DNA detection, and 40.0% (52/130) women were confirmed to infect HPV by HPV E6/E7 mRNA detection, there was no significant difference between the two results (chi2 = 24.5, P < 0.05). The sensitivity, specificity, positive predictive value, negative predictive value of HC2-HPV-DNA detection were 90.1%, 22.1%, 37.4% and 82.6%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value of HPV E6/E7 mRNA detection were 65.9%, 73.3%, 55.8% and 80.8%, respectively.

CONCLUSIONIn clinical cervical screening project of single time, the combination of HC2-HPV-DNA detection and HPV E6/E7 mRNA detection wick take on more potential value than applying each of them alone. RNA;

Adult ; Alphapapillomavirus ; genetics ; isolation & purification ; DNA, Viral ; genetics ; Female ; Genetic Techniques ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; genetics ; Papillomavirus Infections ; virology ; RNA, Viral ; genetics ; Vaginal Smears ; Young Adult

Human papilloma virus (HPV) is believed to be an essential factor for the development of cervical cancer. Early diagnosis and treatment of cervical intraepithelial neoplasia can effectively inhibit the future progression. HPV late 1 protein possesses epitope that can identify and adhere to host cells, and thus may play an important role in HPV infection and cervical carcinogenesis.
Capsid Proteins ; Cervix Uteri ; metabolism ; virology ; Female ; Humans ; Oncogene Proteins, Viral ; Papillomavirus Infections ; complications ; Uterine Cervical Neoplasms ; virology

OBJECTIVETo evaluate the immune potency of recombinant adenovirus combined with rAAV1 vector expressing HPV16L1 protein in mice.

METHODSThe rAdV and rAAV1 vector containing codon-modified HPV16L1 gene was constructed using Admax and AAVmax packaging system respectively. C57 BL/6 mice were immunized with purified rAdV and rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based HPV16 pseudovirus.

RESULTSIntramuscular immunization by rAAV1-mod. HPV16L1 or combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum than that of other groups. The titer of neutralizing antibody of intranasal groups is significantly lower than that of intramuscular group, although the prime-boost strategy using in intranasal group was effective to enhance the specific humoral immunity.

CONCLUSIONThe rAAV1-mod. HPV16L1 combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum through intramuscular route than that of other groups at the 16th week after the first immunization.

Adenoviridae ; genetics ; immunology ; Adenoviridae Infections ; immunology ; Animals ; Antibodies, Viral ; immunology ; Dependovirus ; genetics ; immunology ; Immune System Phenomena ; Immunization ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Fusion ; immunology ; Oncogene Proteins, Viral ; immunology ; Recombinant Proteins ; genetics ; immunology