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

OBJECTIVETo investigate the relationship between the human papillomavirus (HPV) and esophageal carcinoma in Baoding City of Hebei Province.

METHODSWe detected HPV DNA in 42 formalin-fixed and paraffin-embedded tissues from surgically resected esophageal carcinomas from Baoding City of Hebei Province, by PCR with the general primer set of GP5 + /6 + for HPV L1 gene and type-specific primer sets for HPV16 and 18 as well.

RESULTS37 from 42 esophageal carcinoma samples were HPV positive and the rate was 88.1%. Among the samples detected, 19 were HPV16 E6 positive and rate was 45.2%, eight were HPV18 E6 positive and rate was 19.0%.

CONCLUSIONThe high rate of HPV in the esophageal carcinoma samples suggested that HPV plays an etiologic role in the development of esophageal cancer in Baoding City of Hebei Province.

Adult ; Aged ; Carcinoma ; virology ; China ; Esophageal Neoplasms ; virology ; Female ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 18 ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Papillomavirus Infections ; virology

Objectives: To analyze the type and distribution characteristics of human papillomavirus (HPV) infection along with cervical cytology in middle-aged and elderly women in Guangxi and to provide a basis for the prevention and treatment of cervical cancer in elderly women. Methods: 21 subtypes of HPV and cervical cytology of women over 45-year-old visiting the First Affiliated Hospital of Guangxi Medical University from January 2019 to December 2020 were collected. They were divided into two groups by age, 45-64 years group and over 65 years group. The HPV, HR-HPV, and multiple HPV infection prevalence were analyzed, as well as HPV genotypes, the age distribution of HPV infection rate, and cervical cytology. Results: A total of 6 657 eligible women were included. 6 238 women were in the 45-64 years group, with a HPV prevalence about 20.86% (1 301), while 419 women were in the over 65 years group, with a HPV prevalence about 32.94% (138). The age-associated HPV and HR-HPV prevalence increased with the age, peaking at the age group of 70-74 years (P<0.001). The most prevalent genotype was HPV52, and the infection rate was 5.3% (353), followed by HPV16 and HPV 58, about 4.63% (308) and 3.08% (205) respectively. The majority cytology of HPV-positive middle-aged and elderly women was normal. 8.70% (88) of them were ASC-US, 6.52% (66) for HSIL, 4.55% (46) for LSIL, and 2.96% (30) for ASC-H, and 0.10% (1) for SCC. Compared to middle-aged women, elderly women had a lower negative cytology rate, 69.79% (67) vs. 77.95% (714), but a higher HSIL rate, 13.54% (13) vs. 5.79% (53) (P<0.05). Conclusions: HPV and HR-HPV prevalence of elderly women in a medical center of Guangxi are higher than those of middle-aged women. The most prevalent genotype is HPV16 in elderly women, followed by HPV52 and HPV58.
Aged ; China/epidemiology* ; Female ; Hospitals ; Human papillomavirus 16 ; Humans ; Male ; Middle Aged ; Papillomaviridae/genetics* ; Papillomavirus Infections/genetics* ; Uterine Cervical Neoplasms

This study was aimed to identify pET21b-HPV16E2/BL21(DE3) strain and to optimize the expression of human papillomavirus type 16 (HPV16) E2 protein by orthogonal analysis. Four influence factors on two levels were selected to increase the target protein quantity. The four factors were induction time, induction temperature, inductor concentration and cell density. The quantity of HPV16 E2 protein was used as the evaluation parameter. Induced by IPTG, HPV16 E2 protein was analyzed by SDS-PAGE and Western Blot. Target protein was analyzed by GIS imaging system to quantify the protein level. SPSS13. 0 software was applied to analyze the result. Data showed that the expression strain pET211rHPV16 E2/BL21(DE3) was identified correctly. HPV16 E2 protein expressed mainly at insoluble form. The 42KD protein band was identified by SDS-PAGE and Western blot. Orthogonal test was applied on influence factor analysis and expression optimization successfully. Main influence factors were inductor concentration and induction temperature. The optimimum condition of maximum expression quantity was 37 degrees C, 7h, 1.0 mmol/L IPTG and OD600 1.0. In this experiment, orthogonal test could not only be used to analyze the influential factors and promote the target protein expression, but also be used to provide a better experiment method for molecular biological study.
DNA-Binding Proteins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; metabolism ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus Infections ; virology ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo evaluate the diagnostic applicability of human papillomavirus (HPV) liquid chip assay which is based on Luminex XMAP System, and perform a HPV epidemiologic study with the liquid chip in women of Shandong province.

METHODSTo detect HPV genotypes on a 96-well plate with the liquid chip which can simultaneously detect and identify 26 common HPV genotypes in a total of 2925 cervical scrapes obtained from gynecological outpatients as well as to analyze the relationship between HPV types and different cervical diseases by studying the distribution of HPV genotypes and pathologic diagnosis.

RESULTSAmong 639 cases who performed pathologic/cytological and histological diagnoses, 184 cases are in group of normal cytology, 266 cases in group of, 77 cases in group of cervical intra-epithelial neoplasia (CIN) I, 7 cases in group of CIN I - II, 46 cases in group of CIN I - II, 46 cases in group of CIN I - II and 13 cases in group of cervical cancer. The overall incidence of HPV in our samples is 36.0% (1054/2925) and 23 types of all 26 types on liquid chip are found. The most common genotypes found are HPV-16 (26.75%), HPV-52 (25.75%), HPV-58 (10.47%), HPV-18 (8.87%) and HPV-11 (6.94%). Among all the positive types, 87.32% are high-risk HPV and 13.68% are low-risk HPV genotypes. Both single and multiple types are easily identified, showing 66.22% ( n = 698) single type and 33.78% ( n = 356) multiple types. Of all the 1054 HPV-positive cases, 261 (24.8%) is occupied by women 21 to 25 years of age and progressively lower by older age groups, reaching 4.9% by women between 51 to 67 years old. The incidence of HPV in our samples is 23.37%, 33.08%, 54.54%, 57.14%, 82.61%, 91.30% and 100% for normal cytology, inflammation,CIN I ,CIN I - II, CIN II ,CIN III, and carcinomas specimens, respectively. Infections with more that one virus are common, accounted for 4.89%, 7.14%, 18.18%, 28.57%, 41.30%, 43.37% and 38.46% for normal cytology, inflammation, CIN I, CIN I - II, CIN II, CIN III, and carcinomas specimens, respectively. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of HPV liquid chip assay for detecting all cases of CIN II, III are 88.57%, 76.63%, 68.89% and 92.16% respectively. Conclusion The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31. The HPV-positive rate increased along with the increase of grading on cervical lesions. There are more younger women among all the HPV-positive ones. Multiplex HPV genotyping by liquid chip appears to be highly suitable for diagnostic screening as well as the conduction of large-scale epidemiological studies.

Adolescent ; Adult ; Aged ; Cervical Intraepithelial Neoplasia ; epidemiology ; virology ; China ; epidemiology ; Female ; Gammapapillomavirus ; classification ; genetics ; Genotype ; Human papillomavirus 11 ; classification ; genetics ; Human papillomavirus 16 ; classification ; genetics ; Human papillomavirus 18 ; classification ; genetics ; Human papillomavirus 6 ; classification ; genetics ; Humans ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Papillomaviridae ; classification ; genetics ; Papillomavirus Infections ; epidemiology ; virology ; Uterine Cervical Neoplasms ; epidemiology ; virology ; Young Adult

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 analyze how the infection of human papillomavirus 16 (HPV16) affects expression of DNA polymerase beta (DNA polB) with the aim of probing the mechanism of over-expression of DNA polB in human cancers.

METHODSFour fragments of human DNA polB promoter were amplified and constructed into luciferase reporter vector pGL-Basic, generating pGL-BP, pGL-BMH, pGL-BMS, and pGL-BAT constructs respectively, and co-transfected with HPV16 or HPV6 into Hep2 cells. Luciferase activity was assayed 48 hours after transfection. Semi-quantitative RT-PCR was used to measure mRNA expression of endogenous DNA polB. Immunohistochemistry and in situ hybridization were used to analyze DNA polB expression and HPV16 or HPV6 infection in 38 cases of cervical lesions respectively.

RESULTSWith co-transfection of HPV16 and DNA polB promoter-driving reporters into Hep2 cells, pGL-BP reporter in full-length DNA polB promoter presented markedly elevated luciferase activities (P < 0.05). However, the other three mutant reporters: pGL-BMH, pGL-BMS, and pGL-BAT, generated no reporting activities in the presence of HPV16 (P > 0.05). On the contrary, all of polB promoter reporters were little stimulated in co-transfection of HPV6 (P > 0.05). The transfection of HPV16 could enhance the endogenous polB mRNA expression compared with that of HPV6 (3.42 vs. 0.80, P < 0.05). The DNA polB expression was found in 8 of 10 HPV16-positive cervical intraepithelial neoplasia grade III (CIN III) cases, while was only found in 3 of 11 HPV6-positive condyloma accuminatum cases, but was negative in all chronic cervicitis cases. The correlation of DNA polB expression with HPV16 infection in cervical lesions was significant (P < 0.05).

CONCLUSIONSHPV16 is able to specifically stimulate the expression of DNA polB in human epithelial cells through interaction with the core upstream regulatory sequences of DNA polB promoter. Over-expression of DNA polB might be an explanation for the molecular mechanism underlying HPV-related human cancers.

Cell Line ; DNA Polymerase beta ; genetics ; metabolism ; Female ; Gene Expression Regulation, Enzymologic ; Human papillomavirus 16 ; genetics ; metabolism ; Human papillomavirus 6 ; genetics ; metabolism ; Humans ; Papillomavirus Infections ; Promoter Regions, Genetic ; Uterine Cervical Neoplasms ; genetics ; pathology ; virology

BACKGROUND: We evaluated the performance of various commercial assays for the molecular detection of human papillomavirus (HPV); the recently developed AdvanSure HPV Screening real-time PCR assay (AdvanSure PCR) and the Abbott RealTime High Risk HPV PCR assay (Abbott PCR) were compared with the Hybrid Capture 2 HPV DNA Test (HC2). METHODS: All 3 tests were performed on 177 samples, and any sample that showed a discrepancy in any of the 3 tests was genotyped using INNO-LiPA HPV genotyping and/or sequencing. On the basis of these results, we obtained a consensus HPV result, and the performance of each test was evaluated. We also evaluated high-risk HPV 16/18 detection by using the 2 real-time PCR assays. RESULTS: Among the 177 samples, 65 were negative and 75 were positive in all 3 assays; however, the results of the 3 assays with 37 samples were discrepant. Compared with the consensus HPV result, the sensitivities and specificities of HC2, AdvanSure PCR, and Abbott PCR were 97.6%, 91.7%, and 86.9% and 83.9%, 98.8%, and 100.0%, respectively. For HPV type 16/18 detection, the concordance rate between the AdvanSure PCR and Abbott PCR assays was 98.3%; however, 3 samples were discrepant (positive in AdvanSure PCR and negative in Abbott PCR) and were confirmed as HPV type 16 by INNO-LiPA genotyping and/or sequencing. CONCLUSIONS: For HPV detection, the AdvanSure HPV Screening real-time PCR assay and the Abbott PCR assay are less sensitive but more specific than the HC2 assay, but can simultaneously differentiate type 16/18 HPV from other types.
Adult ; Aged ; Cervix Uteri/pathology/virology ; DNA, Viral/analysis ; Female ; Genotype ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; Middle Aged ; Papillomaviridae/*genetics/isolation & purification ; Papillomavirus Infections/*diagnosis/pathology/virology ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Young Adult

OBJECTIVE: Infection with high-risk genotypes of human papillomavirus (HR-HPV) is the major cause of invasive cervical cancers. HPV-16 and HPV-18 are known to be responsible for two-thirds of all invasive cervical carcinomas, followed by HPV-45, -31, and -33. Current guidelines only differentiate HPV-16/18 (+) by recommending direct colposcopy for treatment. We tried to evaluate whether there are differences in risk among 12 non-16/18 HR-HPV genotypes in this study. METHODS: The pathology archive database records of 1,102 consecutive gynecologic patients, who had results for cervical cytology and histology and for HPV testing, as determined by HPV 9G DNA chip, were reviewed. RESULTS: Among the 1,102 patients, 346 were non-16/18 HR-HPV (+) and 231 were HPV-16/18 (+). We calculated the odds ratios for ≥cervical intraepithelial neoplasia 2 (CIN 2) of 14 groups of each HR-HPV genotype compared with a group of HR-HPV (–) patients. Based on the odds ratio of each genotype, we divided patients with non-16/18 HR-HPV genotypes (+) into two groups: HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+). The age-adjusted odds ratios for ≥CIN 2 of the HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+) groups compared with a HR-HPV (–) group were 11.9 (95% CI, 7.6 to 18.8; p<0.001) and 2.4 (95% CI, 1.4 to 4.3; p<0.001), respectively, while that of the HPV-16/18 (+) group was 18.1 (95% CI, 11.6 to 28.3; p=0.003). CONCLUSION: The 12 non-16/18 HR-HPV genotypes can be further categorized (HPV-31/33/35/45/52/58 vs. HPV-39/51/56/59/66/68) by risk stratification. The HPV-31/33/35/45/52/58 genotypes might need more aggressive action. Large scale clinical trials or cohort studies are necessary to confirm our suggestion.
Adult ; Cervical Intraepithelial Neoplasia/*virology ; Colposcopy ; DNA, Viral/analysis ; Female ; *Genotype ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; Middle Aged ; Papanicolaou Test ; Papillomaviridae/*genetics ; Papillomavirus Infections/*virology ; Risk Factors ; Uterine Cervical Neoplasms/virology ; Vaginal Smears