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

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

Objective: To investigate the correlation between methylation in human papillomavirus 16 (HPV16) long control region (LCR) and cervical intraepithelial neoplasia grade ≥2 (CIN2+). Methods: The literature retrieval was conducted by using the databases of PubMed, Embase, Cochrane Library, Web of Science, CNKI, Wanfang data and Weipu according to the inclusion and exclusion criteria, and the retrieval period was from the establishment of the databases to February 27^th, 2022. Software RevMan 5.3 and Stata 15.1 were used for Meta-analysis. Results: A total of 17 literatures were included involving 1 421 subjects. Results of Meta-analysis showed that OR of the correlation between methylation of HPV16 LCR and CIN2+ was 1.56 (95%CI: 0.70-3.47). Subgroup analysis showed that methylation of the 5' terminal, enhancer and promoter regions were not associated with CIN2+, while in four E2 binding sites (E2BS), the methylation of E2BS1, E2BS3 and E2BS4 increased the risk of CIN2+, with the ORs of 3.92 (95%CI: 1.92-7.99), 10.50 (95%CI: 3.67-30.04) and 3.65 (95%CI: 1.58-8.41), respectively. However, subgroup analysis on E2BS2 was not performed due to the limitation of the number of literatures. According to the different sources of population, the risk of CIN2+ in Chinese population was associated with methylation of HPV16 LCR (OR=2.14, 95%CI: 1.31-3.50). There was a correlation between the risk of CIN2+ and HPV16 LCR methylation in the population with pyrosequencing of HPV16 LCR, and OR was 1.75 (95%CI: 1.03-2.98). Conclusion: The risk of CIN2+ is correlated with the methylation of E2BS in HPV16 LCR, which can be used as potential biomarkers.
Female ; Humans ; Methylation ; Human papillomavirus 16/genetics* ; Asian People ; Uterine Cervical Neoplasms ; Uterine Cervical Dysplasia

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

Cell Line, Transformed ; Cell Transformation, Viral ; DNA, Viral ; genetics ; Epithelial Cells ; cytology ; virology ; Human papillomavirus 16 ; genetics ; Humans ; Plasmids

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

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.
Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

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

A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.
Colorimetry ; methods ; DNA Primers ; chemistry ; genetics ; Genotype ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 6 ; genetics ; isolation & purification ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods ; Papillomavirus Infections ; virology