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

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.
Cancer Vaccines ; biosynthesis ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Female ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Papillomavirus Vaccines ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Uterine Cervical Neoplasms ; prevention & control ; Vaccines, DNA ; biosynthesis

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

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

OBJECTIVETo construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression.

METHODSA pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank. The DNA fragment of 1500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene, then cloned into pGEX-KG and transformed into the host E.coli strain JM109. The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degree, the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot.

RESULTSHPV16L1 fusion protein was expressed successfully in the form of inclusion bodies. The molecular weight was 83 kD. Meanwhile, the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol*L(-1) IPTG for 4 h. The fusion protein reacted specifically with antibodies against HPV16L1.

CONCLUSIONThe prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.

Cancer Vaccines ; biosynthesis ; Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Human papillomavirus 16 ; genetics ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

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

Here, we presented a method to bacterially express the major structural protein L1 of Human Papillomavirus type 18 (HPV18) as soluble form. We found that the purified L1 could self-assemble to virus-like particles (VLPs). Further, we investigated the immunogenicity and the induced level of neutralizing antibody using these VLPs. First, the genome of HPV18 was cloned from a patient in Xiamen. It was used as template for PCR amplification of HPV18 L1 gene. The resultant DNA fragment was inserted into expression vector pTrxFus and expressed in Escherichia coli GI724. Second, L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography; and the purified L1 was subjected to self-assembly to form VLPs with the removal of premixed reductant DTT. Finally, the size and morphology of these VLPs was investigated by Dynamic Light Scattering and Transmission Electronic Microscopy as 29.34 nm in hydrated radius and globular particles similar with native HPV18. The half effective dosage (ED50) and maximum level of neutralizing antibody elicitation were measured by vaccinations on mice, rabbit and goat using pseudovirus neutralization cell model. The results showed that the ED50 of HPV18 VLPs is 0.006 microg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 10(7). As a conclusion, we can provide HPV18 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV18.
Animals ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Goats ; Human papillomavirus 18 ; immunology ; isolation & purification ; Mice ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Virion ; genetics ; immunology

OBJECTIVE: To investigate the effect of human papillomavirus types 16E6 on the sensitivity of chemotherapy for cervical carcinoma in different p53 genotype cell lines. METHODS: The apoptosis rates of each group were detected by AO/EB, immunofluorescence and Annexin V/PI stained methods. The expressions of protein HPV16E6 and p53(mt) after the treatments of different concentration of DDP were detected by Western blot. HPV16E6 mRNA in C33A, C33A-E6, C33A-P, and CaSki cell lines under different DDP treatments were detected by RT-PCR. RESULTS: AO/EB and Annexin V/PI stained tests showed that the apoptosis rates of C33A, C33A-E6, C33A-P, and CaSki cells were significantly increased when DDP concentration increased. Western blot showed that the HPV16E6 protein could be detected only in C33A-E6 and CaSki cell lines. The expression of HPV16E6 protein in C33A-E6 and CaSki cell lines gradually decreased and was hardly detected with increased dosage of DDP and the prolonged treatment time (P<0.01), and slightly increased in C33A-E6 and Caski cell lines without the treatment, but there was no significant difference between them (P>0.05). Protein p53(mt) persistently expressed in C33A-E6, C33A, and C33A-P cell lines following the increased dosage of DDP and the prolonged treatment time(P>0.05), while it couldn't be found in CaSki cell line. RT-PCR showed that without DDP intervention, there was no significant difference of HPV16E6 mRNA in C33A-E6 and CaSki cell lines within 24 h.The HPV16E6 mRNA in C33A-E6 cell line expressed much higher than that in CaSki (P<0.05), and HPV16E6 mRNA of 2 cell lines expressed much higher at 48 h than at 24 h (P<0.05).The expression of HPV16E6 mRNA in C33A-E6 and CaSki cell lines gradually decreased with the increased DDP and prolonged treatment time (P<0.01), while there was no significant difference between C33A-E6 and CaSki cell lines under the same DDP concentration (P>0.05). CONCLUSION Effect of HPV16E6 on the sensitivity of chemotherapy for cervical carcinoma cell lines is not markedly related with the different p53 genotype forms(p53(mt)/p53wt ). HPV16E6 may affect the proliferation and sensitivity of chemotherapy in C33A cell line through other mechanism.
Antineoplastic Agents ; pharmacology ; Apoptosis ; genetics ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Female ; Genotype ; Human papillomavirus 16 ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus Infections ; virology ; Repressor Proteins ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Uterine Cervical Neoplasms ; pathology ; virology

OBJECTIVE: To determine the relationship between serum antibody against HPV16 E4 and cervical cancer, and to construct HPV 16 E4 protein expression vector as an antigen to detect its corresponding serum antibody among different populations. METHODS: HPV16 E4 early gene was ligated into pRSET-A expression vector. The constructed plasmids were transformed into BL21 (DE3)cells, and induced to express HPV 16 E4 protein by isopropylthio-beta-D-galactoside (IPTG). The expressed E4 inclusions were denatured, purified through Ni-column, and renatured. After the activity was revealed, antibodies against HPV 16 E4 in the sera from healthy women and patients with chronic cervicitis and cervical cancer were respectively determined by enzyme-linked immunosorbent assay (ELISA) using the fusion protein as the antigen. RESULTS: HPV 16 E4 fusion protein of Mr 15*10(3) was expressed by pRSET-16E4 after IPTG induction. The fusion protein accounted for 30% of the total bacterial proteins and expressed as inclusive body. After purification with Ni-NTA agarose resin, the recombinant protein revealed purity of 95%, and activity of the renatured protein was identified by ELISA. The serum antibody-positive rate of HPV 16 E4 was 10.00%, 39.13% and 28.13%, respectively in 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients. The antibody-positive rate in cervical cancer patients and chronic cervicitis patients were significantly higher than that in healthy women (P<0.01), while the difference between the antibody-positive rate in cervical cancer patients and chronic cervicitis patients was not significant. CONCLUSION HPV 16 E4 protein expressed from pRSET-A/BL21 can be used in serological studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E4 is present in a significantly higher percentage in cervical cancer and chronic cervicitis patients than in healthy women.
Adult ; Aged ; Antibodies, Viral ; blood ; Female ; Genetic Vectors ; Human papillomavirus 16 ; genetics ; immunology ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Papillomavirus Infections ; immunology ; virology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Uterine Cervical Neoplasms ; immunology ; virology ; Uterine Cervicitis ; virology

OBJECTIVETo detect bcl-2 gene expression in Epstein-Barr virus (EBV)-infected human gastric epithelial cell line GES-1 for understanding the role of bcl-2 gene in the carcinogenesis of EBV-associated gastric carcinoma.

METHODSAkata 1061 cells producing recombined EBV carrying neomycin resistance gene (NEOr) was used to mediate the EBV infection of human gastric epithelial cell line GES-1 via close contact, with the empty plasmid pcDNA3-transfected GES-1 cells via lipofectamine method as a control. The EBV-infected and pcDNA3-transfected cells were cloned by limited dilution and the positive clones selected with G418. Immunocytochemical staining was performed to detect the expressions of EBNA1 and Bcl-2 protein.

RESULTSBcl-2 protein expression was detected in EBV-infected cells but not in the control cells.

CONCLUSIONEBV infection can increase Bcl-2 expression in gastric epithelial cells, and such cell transformation effect of EBV is related to the overexpression of bcl-2 gene.

Cell Line, Transformed ; Cell Transformation, Viral ; Epithelial Cells ; cytology ; metabolism ; virology ; Herpesvirus 4, Human ; Humans ; Immunohistochemistry ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Stomach ; cytology