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1.
ABCD (São Paulo, Impr.) ; 34(4): e1637, 2021. tab, graf
Article in English, Portuguese | LILACS | ID: biblio-1360017

ABSTRACT

RESUMO - INTRODUÇÃO: O papilomavírus humano (HPV) é agente das doenças sexualmente transmissíveis de maior prevalência no mundo que estão associadas ao câncer do colo do útero e canal anal. A ação do HPV na carcinogênese colorretal não está ainda estabelecida. OBJETIVO: Estudar a eventual correlação entre a presença do HPV tipo 16 e a expressão gênica da proteína p16INK4a e da oncoproteína E7 de HPV e de seus níveis no tecido do carcinoma colorretal. METODOS: Estudo retrospectivo caso-controle de 79 doentes com carcinoma colorretal divididos em dois grupos: HPV presente e HPV ausente. Foi realizada reação em cadeia da polimerase (PCR), além da hibridização do tipo dot blot para o HPV 16 e o HPV 18 Amostras do tecido colorretal também foram submetidas ao estudo imuno-histoquimico para avaliar o nível tecidual das proteínas E7 e p16INK4a. RESULTADOS: O HPV foi identificado em 36 (45,6%) casos. Não houve diferença significante entre os grupos quanto ao sexo (p=0,056), idade (p=0,1), localização cólica e/ou retal (0,098) e presença do HPV. A expressão gênica da oncoproteína E7 de HPV estava presente em 3,12% dos casos (p=0,9) e a expressão da proteína p16INK4a foi observada em 46,3% (p=0,27) dos indivíduos com detecção do HPV. CONCLUSÃO: A expressão gênica e os níveis teciduais da oncoproteína E7 e da proteína p16INK4a encontrados nos pacientes positivos para o HPV sugerem a ausência de atividade oncogênica do HPV tipo 16 no carcinoma colorretal.


ABSTRACT - BACKGROUND: Human papillomavirus (HPV) is the agent of the most prevalent sexually transmitted diseases in the world associated with cervix and anal canal cancer. The action of HPV on colorectal carcinogenesis is not yet established. OBJECTIVE: This research aimed to study the possible correlation between the presence of HPV16 and the gene expression of p16INK4a protein and HPV E7 oncoprotein and their levels in colorectal carcinoma tissue. METHODS: A retrospective case-control study of 79 patients with colorectal carcinoma was divided into two groups: HPV-positive and HPV-negative. The polymerase chain reaction was performed, in addition to dot-blot hybridization for HPV16 and HPV18. Colorectal tissue samples were also subjected to immunohistochemical study to assess the tissue level of E7 and p16INK4a proteins. RESULTS: HPV was identified in 36 (45.6%) cases. There was no significant difference between groups regarding gender (p=0.056), age (p=0.1), colic and/or rectal location (0.098), and presence of HPV. Gene expression of HPV E7 oncoprotein was present in 3.12% of cases (p=0.9), and p16INK4a protein expression was observed in 46.3% (p=0.27) of those selected with HPV detection. CONCLUSION: Gene expression and tissue levels of E7 oncoprotein and p16INK4a protein found in HPV-positive patients suggest the absence of HPV16 oncogenic activity in colorectal carcinoma.


Subject(s)
Humans , Female , Colorectal Neoplasms/genetics , Colorectal Neoplasms/virology , Cyclin-Dependent Kinase Inhibitor p16/genetics , Papillomavirus Infections/genetics , Papillomavirus E7 Proteins/genetics , DNA, Viral , Case-Control Studies , Retrospective Studies , Human papillomavirus 16/genetics
2.
Rev. invest. clín ; 72(4): 188-197, Jul.-Aug. 2020. tab, graf
Article in English | LILACS | ID: biblio-1251856

ABSTRACT

ABSTRACT Optimal function of the immune system allows the recognition and elimination of infected and tumor cells. However, these cells can develop mechanisms to evade the cellular immune response. In human papillomavirus (HPV) infection, dysregulation of major histocompatibility complex Class I molecules and other components of the innate immune system promote the survival of infected cells by allowing the infection to persist which, in turn, favors the development of cancer. Further, tumor cells possess inherent mechanisms designed to block the recognition and activation of cytotoxic lymphocytes: particularly, HPV proteins such as E1 and E2 and oncoproteins E5, E6, and E7 that inhibit immune mechanisms and/or stimulate the expression of immunosuppressive cytokines. These mechanisms include a decrease in receptor activation and costimulating molecules on the surface of immune cells, as well as the constitutive expression of molecules that inhibit their function, which allow HPV persistence and tumor progression. Immunotherapy-based therapeutic options are positioned as excellent candidates for the treatment of cervical cancer.


Subject(s)
Humans , Female , Histocompatibility Antigens Class I , Uterine Cervical Neoplasms/immunology , Oncogene Proteins, Viral , Papillomavirus Infections/complications , Papillomavirus Infections/therapy , Uterine Cervical Neoplasms/virology , Papillomavirus E7 Proteins , Immunotherapy
3.
Journal of Gynecologic Oncology ; : e88-2019.
Article in English | WPRIM | ID: wpr-764572

ABSTRACT

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


Subject(s)
Female , Humans , Antibody Formation , Biopsy , Carcinoma in Situ , Uterine Cervical Dysplasia , Cervix Uteri , Cohort Studies , Immunity, Humoral , Immunization , Lacticaseibacillus casei , Papillomavirus E7 Proteins , Papillomavirus Vaccines , Uterine Cervical Neoplasms
4.
Braz. j. infect. dis ; 21(3): 248-254, May-June 2017. tab
Article in English | LILACS | ID: biblio-839224

ABSTRACT

ABSTRACT Objective: To evaluate the association between p53 polymorphisms and human papillomavirus (HPV) E6/E7 mRNA expression. Methods: We analyzed 175 cervical samples from women aged 16-69 years old who were tested for HPV E6/E7 mRNA expression (NucliSENS® EasyQ® HPV). The samples were divided into three groups: positive (n = 75) those with positive HPV E6/E7 mRNA expression and positive high-risk HPV Hybrid Capture (HR-HC) test; negative (n = 52) those with negative HPV E6/E7 mRNA expression and positive HR-HC; and control (n = 48) those with negative HPV E6/E7 mRNA expression and negative HR-HC. The p53 polymorphisms at codons 11, 72, and 248 were evaluated through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The frequency of the arginine/arginine homozygous genotype at codon 72 was significantly higher in the positive (49.3%) than in the negative (32.7%) and control groups (20.8%, p = 0.002*). The frequency of the arginine allele was also significantly higher in the positive (67.3%) than in the negative (53.8%) and control groups (38.5%, p < 0.001*). The arginine/arginine homozygous genotype was significantly associated with positive HPV E6/E7 mRNA expression (positive group) compared with negative and control groups (odds ratio: 2.633; 95% CI, 1.399-4.954, p = 0.003). The frequency of arginine/arginine homozygous genotype at codon 72 remained significantly more frequent in the positive group of women aged ≥30 years than in the other two groups. Conclusion: The presence of the p53 arginine/arginine homozygous genotype at codon 72 was significantly associated with the positive HPV E6/E7 mRNA expression.


Subject(s)
Humans , Female , Adolescent , Adult , Middle Aged , Aged , Young Adult , Papillomaviridae/genetics , RNA, Messenger/metabolism , Oncogene Proteins, Viral/genetics , Uterine Cervical Dysplasia/virology , Papillomavirus Infections/virology , Papillomavirus E7 Proteins/genetics , Arginine/genetics , Polymorphism, Restriction Fragment Length , Codon , RNA, Viral , Uterine Cervical Neoplasms/virology , Polymerase Chain Reaction , Tumor Suppressor Protein p53/genetics , Genotype
5.
Chinese Journal of Biotechnology ; (12): 683-692, 2016.
Article in Chinese | WPRIM | ID: wpr-337431

ABSTRACT

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD₅₀) of LM4△hly::E7 strain was 3.863×10⁹ CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.


Subject(s)
Animals , Mice , Cancer Vaccines , Allergy and Immunology , Listeria monocytogenes , Mice, Inbred C57BL , Papillomavirus E7 Proteins , Allergy and Immunology , Papillomavirus Infections , Plasmids , Recombinant Fusion Proteins , Allergy and Immunology , Vaccines, Attenuated , Allergy and Immunology , Viral Vaccines , Allergy and Immunology
6.
Iranian Journal of Cancer Prevention. 2015; 8 (1): 18-23
in English | IMEMR | ID: emr-159983

ABSTRACT

Cervical cancer has been shown to be highly associated with human papillomavirus [HPV] infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore potent targets for therapeutic genetic vaccination. In the present study, it was investigated the potential effect of HPV-16 E6, E7 and L1 co-administration to activate specific cytotoxic T lymphocytes in tumor mice models. The HPV-16 E6, E7 and L1 genes from Iranian isolate were separately inserted into the mammalian expression vector, pcDNA3, to construct the DNA vaccine candidates. Tumor-bearing Animals [C57BL/6 mice] were immunized with the vaccine candidate; then, Lymphocyte Proliferation Assay [LPA] and relative tumor volume measurements were carried out in order to examine the immunological effects of the vaccine. Obtained results showed that co-administration of the HPV-16 E6, E7 and L1 DNA induced HPV-16 specific cellular immune responses and also protected against TC-1-induced tumor in vivo compared with negative controls. The results showed that mixed delivery systems might be valuable to improve the magnitude of the induced immune responses and confirmed therapeutic effects of HPV-16 E6, E7 through cytotoxic T lymphocyte induction and illustrate the new promising role for HPV-16 L1 CTL epitopes as a suitable CTL inducer


Subject(s)
Animals, Laboratory , Vaccines, DNA , Immunity, Cellular , Oncogene Proteins, Viral , Repressor Proteins , Papillomavirus E7 Proteins , Capsid Proteins , Human papillomavirus 16 , Mice
7.
Chinese Journal of Pathology ; (12): 884-888, 2015.
Article in Chinese | WPRIM | ID: wpr-278507

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.</p><p><b>METHODS</b>Both SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively.</p><p><b>RESULTS</b>Cyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells.</p><p><b>CONCLUSION</b>Squamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.</p>


Subject(s)
Female , Humans , Carcinoma, Squamous Cell , Metabolism , Virology , Cell Line, Tumor , Uterine Cervical Dysplasia , Metabolism , Virology , Cyclin D1 , Genetics , Metabolism , Human papillomavirus 16 , Immunohistochemistry , Papillomavirus E7 Proteins , Genetics , Promoter Regions, Genetic , RNA Interference , RNA, Messenger , RNA, Small Interfering , Transfection , Uterine Cervical Neoplasms , Metabolism , Virology
8.
Chinese Journal of Biotechnology ; (12): 566-576, 2015.
Article in Chinese | WPRIM | ID: wpr-240618

ABSTRACT

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.


Subject(s)
Animals , Mice , Capsid Proteins , Codon , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetic Vectors , Human papillomavirus 16 , Neoplasms, Experimental , Oncogene Proteins, Viral , Papillomavirus E7 Proteins , Papillomavirus Vaccines , Therapeutic Uses , Recombinant Fusion Proteins
9.
Cell Journal [Yakhteh]. 2013; 15 (2): 176-181
in English | IMEMR | ID: emr-127542

ABSTRACT

For immunotherapy of human papillomavirus [HPV] -16-associated cervical cancers the E7 protein is considered a prime candidate. However it is a poor inducer of cytotoxic T-cell response, when being used as a singular antigen in protein vaccination. Hence, in this study we focused on the utilization of a vaccine delivery system for prevention or treatment of cervical cancer. In this experimental study, we designed and evaluated a novel fusion protein comprising HPV16 E7 antigen fused to Shiga toxin B-subunit [STxB] as both an antigen vector and an adjuvant. Then we designed two preventive and therapeutic tumor models to investigate the prevention and inhibition of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-STxB or E7 without any adjuvant. We demonstrated that prophylactic immunization of E7-STxB protected mice against TC-1 cells. Also in the therapeutic model, E7-STxB inhibited TC-1 tumor growth inlungs. The results were significant when compared with the immunization of E7 singularly. We concluded that immunization with the E7-STxB protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells. This research verifies the clinical applications and the future prospects of developing HPV16 E7 therapeutic vaccines fused to immunoadjuvants


Subject(s)
Animals, Laboratory , Papillomavirus E7 Proteins , Mice , Shiga Toxin , Shigella dysenteriae , Uterine Cervical Neoplasms , Immunization
10.
Chinese Journal of Integrated Traditional and Western Medicine ; (12): 1545-1551, 2013.
Article in Chinese | WPRIM | ID: wpr-231648

ABSTRACT

<p><b>OBJECTIVE</b>To explore the in vitro inhibitive effect and underlying mechanisms of Brucea Javanica oil emulsion (BJOE) on human papilloma virus (HPV) type 16 infected cells.</p><p><b>METHODS</b>The 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).</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSIONS</b>BJOE 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.</p>


Subject(s)
Female , Humans , Apoptosis , Brucea , Chemistry , Cell Line, Tumor , Drugs, Chinese Herbal , Pharmacology , Human papillomavirus 16 , Virulence , Oncogene Proteins, Viral , Metabolism , Papillomavirus E7 Proteins , Metabolism , Papillomavirus Infections , Plant Oils , Pharmacology , Repressor Proteins , Metabolism
11.
Chinese Journal of Oncology ; (12): 491-496, 2013.
Article in Chinese | WPRIM | ID: wpr-267514

ABSTRACT

<p><b>OBJECTIVE</b>To develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity.</p><p><b>METHODS</b>The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay.</p><p><b>RESULTS</b>Sequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group.</p><p><b>CONCLUSIONS</b>The fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.</p>


Subject(s)
Animals , Female , Mice , Cancer Vaccines , Allergy and Immunology , Capsid Proteins , Genetics , Allergy and Immunology , Cell Transformation, Neoplastic , Cloning, Molecular , Codon , Immunoglobulin G , Metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , NIH 3T3 Cells , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Papillomaviridae , Papillomavirus E7 Proteins , Genetics , Allergy and Immunology , Papillomavirus Vaccines , Allergy and Immunology , Plasmids , Point Mutation , Random Allocation , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Transfection , Vaccines, DNA , Allergy and Immunology
12.
Journal of Biomedical Engineering ; (6): 1102-1107, 2013.
Article in Chinese | WPRIM | ID: wpr-259759

ABSTRACT

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.


Subject(s)
Female , Humans , Cancer Vaccines , Genetics , Capsid Proteins , Genetics , Oncogene Proteins, Viral , Genetics , Papillomavirus E7 Proteins , Genetics , Papillomavirus Vaccines , Recombinant Proteins , Genetics , Allergy and Immunology , Uterine Cervical Neoplasms , Vaccines, DNA
13.
Chinese Journal of Virology ; (6): 554-559, 2012.
Article in Chinese | WPRIM | ID: wpr-340006

ABSTRACT

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.


Subject(s)
Animals , Female , Humans , Mice , Cell Line , Virology , Human papillomavirus 31 , Genetics , Metabolism , Oncogene Proteins, Viral , Genetics , Metabolism , Papillomavirus E7 Proteins , Genetics , Metabolism , Papillomavirus Infections , Virology , Recombinant Proteins , Genetics , Metabolism , Transfection
14.
Chinese Journal of Virology ; (6): 645-651, 2012.
Article in Chinese | WPRIM | ID: wpr-339991

ABSTRACT

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.


Subject(s)
Adult , Female , Humans , Middle Aged , Base Sequence , China , Genetic Variation , Human papillomavirus 16 , Classification , Genetics , Molecular Sequence Data , Mutation , Oncogene Proteins, Viral , Genetics , Papillomavirus E7 Proteins , Genetics , Papillomavirus Infections , Virology , Phylogeny , Repressor Proteins , Genetics
15.
Chinese Journal of Oncology ; (12): 810-815, 2012.
Article in Chinese | WPRIM | ID: wpr-307288

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.</p><p><b>METHODS</b>The HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.</p><p><b>RESULTS</b>The codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.</p><p><b>CONCLUSIONS</b>The data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.</p>


Subject(s)
Animals , Female , Humans , Mice , Adjuvants, Immunologic , Pharmacology , Cancer Vaccines , Allergy and Immunology , Therapeutic Uses , Capsid Proteins , Genetics , Allergy and Immunology , Metabolism , Cell Line, Tumor , Cell Proliferation , Codon , Escherichia coli , Allergy and Immunology , Metabolism , Immunization , Methods , Immunotherapy , Methods , Mice, Inbred C57BL , Neoplasm Transplantation , Oligodeoxyribonucleotides , Allergy and Immunology , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Metabolism , Papillomavirus E7 Proteins , Genetics , Allergy and Immunology , Metabolism , Papillomavirus Vaccines , Allergy and Immunology , Therapeutic Uses , Plasmids , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , Repressor Proteins , Genetics , Allergy and Immunology , Metabolism
16.
Chinese Journal of Virology ; (6): 51-56, 2012.
Article in Chinese | WPRIM | ID: wpr-354772

ABSTRACT

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.


Subject(s)
Animals , Female , Mice , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Mice, Inbred C57BL , Papillomavirus E7 Proteins , Genetics , Polymerase Chain Reaction , Recombinant Proteins
17.
Invest. clín ; 51(2): 193-208, jun. 2010.
Article in Spanish | LILACS | ID: lil-574073

ABSTRACT

Se examinó la historia de la relación entre la infección con el VPH, las lesiones intraepiteliales y el cáncer del cuello uterino. Inicialmente los hallazgos fueron descritos en Maracaibo (1971), luego en México en 1973 y posteriormente los estudios sobre la ultraestructura e inmunohistoquímica de este virus y su importancia en la génesis del cáncer cervical. Se describió la ultraestructura de los viriones y sus diferentes proteínas señalando el rol de ellas en la incorporación del genoma viral a los queratinocitos del cérvix. La cubierta glicoproteica de los queratinocitos ha sido objeto de estudios y se señaló la importancia de la misma durante la infección con el VPH y su relación con p16, los antígenos de grupos sanguíneos y alteraciones tempranas en diferentes genes, las que conllevan cambios en el ciclo celular con pérdida de la heterocigosis, fenómenos que estimulados por la infección con el VPH de alto riesgo, conducen al cáncer del cuello uterino.


The history on the relationship of VPH infection and cervical cancer was examined. Findings were initially reported in Maracaibo(1971), later in Mexico(1973) and thereafter several studies on the ultrastructure and immunohistochemistry of VPH infection and its role on cervical cancer were described. The ultrastructural findings of viral particles of HPV and their proteins, as well as their role in the incorporation of the viral genome to the human cervical cells were also described. Glycoproteins on the surface of cervical cells were reviewed and their importance on HPV infection was related to p16, blood group antigens and early genetic changes in the cell cycle with loss of heterozigocity, all of which, stimulated by the high risk HPV infection lead to cervical cancer.


Subject(s)
Humans , Female , Immunohistochemistry , Papillomavirus E7 Proteins , Papillomavirus Infections , Uterine Cervical Neoplasms
18.
Chinese Journal of Experimental and Clinical Virology ; (6): 24-26, 2010.
Article in Chinese | WPRIM | ID: wpr-316977

ABSTRACT

<p><b>OBJECTIVE</b>To study the distribution of human papillomavirus type 16 (HPV16) variants and their clinical significance in Han women with Cervical Intraepithelial Neoplasia (CIN).</p><p><b>METHODS</b>Randomly making a collection of DNA samples of cervical cells from 77 Han out-patients infected with HPV16, PCR amplification of HPV16 DNA fragments containing E6 and E7 genes and sequenced. To study the HPV16 variants types in these out-patients and explore the relationship between the HPV16 variants and CIN by comparing the E6 genes sequenced with the reference strains downloaded from the GenBank. RESULTS Among 77 patients, the minimum age is 21 years old, the maximum age is 56 years old, and the average age is 36.39 +/- 6.86 years old. 61 patients (accounting for 79.2%) were diagnosed as CIN II and higher grade lesions while 16 patients (accounting for 20.8%) as CIN I. In this research, only European variant and Asian variant were found by Parsimony analyses of the sequences. There are 38 Asian variants and 39 European variants. With Chi2 test, Chi2 = 0.0034, P = 0.9535 > 0.05, it suggested that there was no enough evidence to support Asian- and European-variants had the different risk in the cause of cervical intraepithelial neoplasia and cervical cancer.</p><p><b>CONCLUSION</b>It was not found Asian- and European-variants of HPV16 had different effect on the cervical cancer, but found only two major variants-Asian- and European-variants in Han people in this research. So we have reason to speculate that there are two major HPV16 variants (Asian- and European-variants) in China's Han women, while other variants, especially high cancer-causing Asian/American variant are not common.</p>


Subject(s)
Adult , Female , Humans , Middle Aged , Young Adult , Uterine Cervical Dysplasia , Genetic Variation , Human papillomavirus 16 , Classification , Genetics , Molecular Sequence Data , Oncogene Proteins, Viral , Genetics , Papillomavirus E7 Proteins , Genetics , Papillomavirus Infections , Pathology , Virology , Phylogeny , Repressor Proteins , Genetics , Risk Factors , Uterine Cervical Neoplasms , Pathology , Virology
19.
Chinese Journal of Virology ; (6): 368-372, 2010.
Article in Chinese | WPRIM | ID: wpr-286110

ABSTRACT

To study the gene variation and the distribution of HPV16 variant in Hubei, China, DNA was extracted from cervical cancer tissue samples. The E6 and E7 genes of HPV16 were amplified and the PCR products were sequenced using E6- and E7-specific primers. Fortyseven cases were found mutations at nucleotide position 178 of HPV16 E6 gene in 80 cervical cancer samples. This mutation resulted in amino acid change from Asp to Glu. The rate of mutation at nucleotide position 178 of E6 gene was 58. 75%. Twenty two cases were found mutations at nucleotide position 647 of HPV16 E7 gene in 31 cervical cancer samples. This mutation resulted in amino acid change from Asn to Ser. The rate of mutation was 70.97%. These results showed that mutations at nucleotide position 178 of E6 gene, nucleotide position 647 of E7 gene of HPV16 in cerveical cancer samples were prevalent in Hubei, China. Phylogenetic analysis showed that Asian (As) variants of HPV16 are predominated in Hubei, China. European (Ep) varinats were also found in Samples in Hubei areas. None of Asian American (AA), African-1 (Af-1), African-2 (Af-2) variants of HPV16 was found in this region. Whether Asian (As) variants of HPV16 are more oncogenic and play a much more important role in the progress of cervical cancer than European (Ep) variants is not clear. More sequences of E6 and E7 gene in CIN and normal cervical tissue samples and study of the function of E6 and E7 protein of these HPV16 variants are needed to adress above question.


Subject(s)
Adult , Female , Humans , Middle Aged , China , Evolution, Molecular , Mutation , Oncogene Proteins, Viral , Chemistry , Classification , Papillomavirus E7 Proteins , Classification , Genetics , Phylogeny , Polymerase Chain Reaction , Repressor Proteins , Chemistry , Classification , Uterine Cervical Neoplasms , Virology
20.
Chinese Journal of Oncology ; (12): 107-110, 2010.
Article in Chinese | WPRIM | ID: wpr-260457

ABSTRACT

<p><b>OBJECTIVE</b>The aim of this study was to analyze the relative distribution and gene variation of HPV16 transforming gene E6, E7 and E5 at different stages of cervical lesions.</p><p><b>METHODS</b>DNA was extracted from tissue samples of 200 patients with cervical lesions, including 124 cervical cancer, 17 CIN grade I and II, 23 CIN grade III and 36 cervicitis. Then HPV16 E6, E7 and E5 genes were amplified, and part of the E6 and E7 PCR products were sequenced using the HPV16 E6 and E7 specific primers.</p><p><b>RESULTS</b>The positive rate of E6 gene in cervicitis, CINI and CINII, CINIII and cervical cancer was 25.0%, 29.4%, 60.9% and 76.6%, respectively. The positive rate of E7 gene was 16.7%, 41.2%, 43.5% and 61.3%, respectively. The positive rate of E5 gene was 5.6%, 5.9%, 30.4% and 40.3%, respectively. HPV16 E6 gene mutations in Nt 178 were found in 47 case from 80 cervical cancer samples, resulting in amino acid change of Asp to Glu. The mutation rate was 58.8%.Otherwise the mutation rate of E6 178 in cervicitis and CIN I approximately III samples was 25.0% and 31.8%. E7 mutations were found in Nt 647 in 21 cervical samples from 30 cervical cancer samples, resulting in amino acid change of Asn to Ser. The mutation rate was 70.0%. The mutation rate of E6 647 in cervicitis and CINI approximately III samples was 35.0% and 40.9%, respectively.</p><p><b>CONCLUSION</b>The positive rate of E6 and E7 increase gradually from cervicitis, CINI and CINII, CINIII to cervical cancer. The rate of E5 is relatively lower than that of E6 and E7 gene in cervical tissue samples. These results show that E6 and E7 gene are highly associated with the progress of cervical cancer and E5 genes are lost in the development of cervical cancer. High frequency mutations of HPV16 E6 and E7 gene in E6 178, E7 647 have been found in cervical cancer samples in Hubei province, China. These results approved that the HPV16 variants prevalent in this area are different from the European and African variants.</p>


Subject(s)
Adult , Female , Humans , Middle Aged , Carcinoma , Metabolism , Virology , Uterine Cervical Dysplasia , Metabolism , Virology , China , Human papillomavirus 16 , Genetics , Oncogene Proteins, Viral , Genetics , Metabolism , Papillomavirus E7 Proteins , Genetics , Metabolism , Papillomavirus Infections , Genetics , Point Mutation , Repressor Proteins , Genetics , Metabolism , Uterine Cervical Neoplasms , Metabolism , Virology , Uterine Cervicitis , Metabolism , Virology
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