1.Optimized expression, preparation of human papillomavirus 16 L2E7 fusion protein and its inhibitory effect on tumor growth in mice.
Yunshui JIANG ; Jianbo LI ; Meng GAO ; Jiao REN ; Sufeng JIN ; Gang CHEN ; Jie WU ; Fangcheng ZHUANG ; Houwen TIAN
Chinese Journal of Biotechnology 2015;31(4):566-576
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
2.Immunogenicity and antitumor efficacy of the recombinant adenovirus expressing E7 and E6 fussion proteins of HPV type 16 in mice
Jiao REN ; Li ZHAO ; Houwen TIAN ; Jian GAO ; Jing FENG ; Zheng PANG ; Xiaobing WU ; Wenjie TAN ; Li RUAN
Chinese Journal of Microbiology and Immunology 2012;32(3):276-280
ObjectiveTo construct one recombinant adenovirus AdE7E6 expressing HPV16 E6 and E7 fusion protein as candidate for HPV16 therapeutic vaccine.MethodsThe codon-optimized E6 and E7 gene,were fused to create one open reading frame,then inserted into adenovirus vector pCD316.A strain of recombinant adenovirus was constructed through homologous recombinant in 293 cells,and identified by PCR and Western blot.Finally,it was employed to study it's immunogenicity and the activity of the tumor growth regression.ResultsThe PCR result showed that E6E7 fusion gene had been integrated in recombinant Ad5 DNA.Western blot test confirmed that the E6E7 fusion protein was highly expressed in 293 cells infected with Ad5E7E6 recombinant adenovirus.The recombinant adenovirus elicited significant E7 specific CD8+ T lymphocyte response in vaccinated mice.These responses could completely prevent the TG-1 tumor cell bearing mice treated with AdE7E6 from developing into tumor.ConclusionThese results suggested that rAd5E7E6 could be a potent vaccine candidate for the treatment of HPV16-associated tumors and their precancerous transformations.
3. Research progress in monkeypox virus detection
Chinese Journal of Experimental and Clinical Virology 2017;31(3):273-276
Monkeypox is a zoonotic disease caused by monkeypox virus. It is similar to human smallpox, although typically much less serious and limited to human-to-human transmission. Smallpox no longer occurs following its worldwide eradication in 1980, whereas monkeypox still occurs sporadically in parts of Africa. To respond to the outbreak of monkeypox correctly, we need to rely on effective method of virus detection. This review presents the development in monkeypox virus detection method in recent years to provide reference for the situation of potential virus spreading into our country and for preventing and acting correctly against bioterrorism.
4.Construction and identification of the replication-deficient recombinant vaccinia virus co-expressing HPV type 16 L1 and L2 proteins.
Liqun HAN ; Jiao REN ; Yu LIANG ; Houwen TIAN ; Huijun ZHI ; Weifeng LUO ; Zhenhua LU ; Lanlan WEI ; Li RUAN
Chinese Journal of Experimental and Clinical Virology 2002;16(3):256-260
OBJECTIVETo generate an HPV16 prophylactic vaccine candidate for cervical cancer.
METHODSHPV16 major capsid protein L1 gene and minor capsid protein L2 gene were amplified using PCR. These genes were mutated by PCR site-directed mutagenesis for removal of sequence motifs (TTTTTNT) which would cause transcription termination when expressed from a vaccinia virus early promoter, then inserted into a vaccinia virus expression vector. A strain replication-deficient recombinant vaccinia virus containing the mutant sequences was obtained through a homologous recombination and identified.
RESULTSThe nucleotide sequence remained the correct amino acid sequence of the L1 and L2 proteins after mutated. Full-length L1 and L2 proteins were generated in cells infected with the recombinant virus. The virus strain propagated at very low titer or could not reproduce in some kinds of cell derived from different human tissues.
CONCLUSIONSThe authors have generated a strain replication-deficient recombinant vaccinia virus expressing HPV16 L1 plus L2 proteins as an HPV16 prophylactic vaccine candidate for cervical cancer.
Capsid ; Capsid Proteins ; genetics ; Cell Line ; Cloning, Molecular ; Female ; Gene Expression ; Genetic Vectors ; Humans ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; prevention & control ; Transfection ; Tumor Virus Infections ; prevention & control ; Uterine Cervical Neoplasms ; virology ; Vaccinia virus ; genetics ; physiology ; Virus Replication
5.Construction of recombinant vaccinia virus co-expressing mutant E6 plus E7 proteins and detection of its immunogenicity and antitumor response.
Huijun ZHI ; Liqun HAN ; Jiao REN ; Houwen TIAN ; Weifeng LUO ; Yu LIANG ; Li RUAN
Chinese Journal of Experimental and Clinical Virology 2002;16(4):341-344
OBJECTIVETo generate a candidate HPV16 vaccine for experimental and therapeutical use for cervical cancer.
METHODSThe mutants of HPV16 early E6 and E7 genes were inserted into a vaccinia virus expression vector. A strain of recombinant vaccinia virus was constructed through homologous recombination.
RESULTSShowed that the mutant E6 and E7 genes were located at TK gene region of vaccinia virus Tiantan strain in a head to head orientation under the control of early/late promoters, H6 and 7.5K respectively. Studies in mice indicated that VmE6E7 could elicit specific antibodies against E6 and E7, and retarded or prevented tumor development in a proportion of C57 BL/6 mice challenged by syngeneic HPV16E6 and E7 transformed tumor cells.
CONCLUSIONSThe success in constructing VmE6E7 provides a basis for the further development of HPV16 therapeutic vaccine.
Animals ; Female ; Genes, Viral ; genetics ; Genetic Vectors ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Mutation ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Recombination, Genetic ; Repressor Proteins ; Transfection ; Vaccinia virus ; genetics ; immunology
6.Modification of HPV type 16 E6 and E7 genes, and analysis of stability and immunogenicity of the modified proteins.
Huijun ZHI ; Liqun HAN ; Jiao REN ; Houwen TIAN ; Weifing LUO ; Yu LIANG ; Li RUAN
Chinese Journal of Experimental and Clinical Virology 2002;16(2):124-127
BACKGROUNDTo select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer.
METHODSE6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity.
RESULTSWestern blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.
Animals ; Antibodies, Viral ; biosynthesis ; Female ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Insertional ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; Vaccinia virus ; immunology ; Zinc Fingers
7.Chemical constituents from Spongia sp., a marine sponge in Xisha Islands
Yuan TIAN ; Bin XU ; Jingfeng WANG ; Houwen LIN ; Lianjuan YANG ; Fan YANG
Journal of Pharmaceutical Practice 2017;35(4):315-320,382
Objective To investigate the chemical constituents in the marine sponge, Spongia sp., collected from the Xisha Islands.Methods The pure chemical components from the petroleum ether extract of Spongia sp.were obtained by repeated column chromatography on silica gel,ODS,Sephadex LH-20 and semi-preparative HPLC.Their structures were determined by spectroscopic analysis and comparison with the reported data.The antifungal activity of those compounds was evaluated by dilution method.Results 9 compounds were isolated and identified,including smenodiol(1),smenospongorine(2),5-epi-smenospongorine(3),dictyoceratin C(4),epi-smenospongidine(5),dictyoceratin A(6),stigmasta-4,6,8(14),22-tetraen-3-one(7),3-oxo-4,6,8(14)-triunsaturated steroids(8),ergosta-4,6,8(14),22-tetraen-3-one(9).Conclusion Compounds 1~9 were isolated from the sponge of genus spongia for the first time.Compound 2、3、5 and 9 exhibited antifungal activities against Candida albicans,Trichophyton mentaqrophytes and Trichophyton rubrum with the MIC values of 12.5~25 μg/ml.
8. Preliminary exploration of replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan
Panpan HUANG ; Li ZHAO ; Jiao REN ; Ying ZHAO ; Li RUAN ; Wenjie TAN ; Houwen TIAN
Chinese Journal of Experimental and Clinical Virology 2018;32(2):119-123
Objective:
To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan.
Methods:
We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected.
Results:
We have expressed and purified vaccinia virus proteins respectively in
9. Generation and characterization of specific monoclonal antibodies against monkeypox virus
Qianqian GUAN ; Li ZHAO ; Jiao REN ; Panpan HUANG ; Huijuan WANG ; Yingzhu CHEN ; Na ZHU ; Wenjie TAN ; Li RUAN ; Houwen TIAN
Chinese Journal of Experimental and Clinical Virology 2017;31(2):153-156
Objective:
To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay.
Methods:
Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in
10. Preparation and characteristic analysis of six influenza A (H7N9) pseudovirus derived from different districts of China
Baoying HUANG ; Shanqin LI ; Xiangrong QI ; Jiao REN ; Jingdong SONG ; Wenjie TAN ; Houwen TIAN ; Li RUAN
Chinese Journal of Experimental and Clinical Virology 2017;31(4):281-286
Objective:
To prepare strains of influenza A (H7N9) pseudovirus derived from different districts of China for vaccine efficacy evaluation.
Methods:
Phylogenetic tree was built based on hemagglutinin (HA) amino acid sequence analyses from 29 influenza A (H7N9) virus strains and 6 influenza A (H7N9) virus strains with HA determinants variation were selected. 293FT cells were co-transfected with plasmid pNL4-3-Luc.R-E-, pVRC-HA and pVRC-NA with codon-optimized hemagglutinin (HA) and neuraminidase (NA) derived from the six influenza A (H7N9) virus strains, respectively. Transmission electron microscopy assay and Western blot analysis were performed to demonstrate morphology and specificity of these particles, luciferase activity assay and hemagglutinin titers detection were used to determine their infectivity and hemagglutinin activity. And finally, pseudovirus-based neutralization assays were evaluated with HA immunized mice serum.
Results:
Six influenza A (H7N9) peseudovirus particles derived from different districts of China were selected and prepared. All of the particles bearing HA and NA were characterized with classic influenza virus morphology, with TCID50 titer ranged from 104TCID50/50 μl to 105TCID50/50 μl and with hemagglutinin activity ranged from 64 to 512. Neutralization efficacies on influenza A/Shanghai/1/2013(H7N9) HA vaccine serum against 100TCID50 dose of these pseudovirus particles indicated their potential application in the vaccine cross-protective evaluation in future.
Conclusions
Six influenza A (H7N9) pseudovirus derived from different districts of China with potential antigenic variation on HA were constructed successfully, established foundation for their further application in vaccine cross-reactive efficacy evaluation.