1.Construction and identification of non-replication recombinant vaccinia virus co-expressing human papillomavirus type 16 L1/L2/E6/E7 proteins.
Wei HUANG ; Hou-wen TIAN ; Jiao REN ; Jiang-tao FAN ; Li ZHAO ; Tao BIAN ; Zhen-hua LU ; Li RUAN
Chinese Journal of Experimental and Clinical Virology 2005;19(3):240-243
OBJECTIVETo generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer.
METHODSHPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified.
RESULTSDNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus.
CONCLUSIONNTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.
Animals ; Blotting, Western ; Capsid Proteins ; genetics ; metabolism ; Cells, Cultured ; Chick Embryo ; Cloning, Molecular ; Female ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomaviridae ; genetics ; immunology ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; immunology ; prevention & control ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; therapeutic use ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Transfection ; Tumor Virus Infections ; immunology ; prevention & control ; virology ; Uterine Cervical Neoplasms ; immunology ; prevention & control ; virology ; Vaccinia virus ; genetics ; Virus Replication
2.Prokaryotic expression and purification of human papillomavirus type 11 L2E7 fusion protein vaccine and its immunnogenicity.
Hui ZHANG ; Li ZHAO ; Jiao REN ; Jian GAO ; Tao BIAN ; Jiang-tao FAN ; Li RUAN ; Xin-qiu CHEN ; Hou-wen TIAN
Chinese Journal of Experimental and Clinical Virology 2007;21(2):156-158
OBJECTIVETo construct the Escherichia coli (E. coli) prokaryotic expression system pET9aHPV11L2E7, purify the fusion protein L2E7 and study the immunnogenicity of the protein.
METHODSThe HPV11 L2, E7 coding region was amplified from condyloma acuminata tissue specimen by PCR. The recombinant plazmid pET9aHPV11L2E7 was established and sequenced. Fusion protein L2E7 (553 amino acids) was expressed in host strain BL21 (DE3plus) by IPTG inducing and identified by using SDS-PAGE and Western blotting. Then L2E7 protein purified with CM column was inoculated to Balb/c mice and its cell-mediated and humoral immunnogenicity was assessed by IFN-gamma enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA).
RESULTSThe E. coli prokaryotic expression system pET9aHPV11L2E7 was established and the purified fusion protein L2E7 was obtained successfully. The mice in vivo experiment indicated that the purified protein L2E7 could induce HPV11E7 specific cell-mediated immune responses and high level HPV L2E7 antibody was detected in serum.
CONCLUSIONThe purified fusion protein L2E7 could induce specific cell-mediated and humoral immune responses. It can be used as a candidate of genital wart immune therapeutic vaccine.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Human papillomavirus 11 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins, Viral ; genetics ; immunology ; isolation & purification ; Papillomavirus Infections ; immunology ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; isolation & purification ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification
3.Expression of the human papillomavirus type 16L/E7 fusion protein in E. coli and observation of its immunogenicity in mice.
Hou-wen TIAN ; Zhen-mei YE ; Zhen-hua LU ; Ren JAO ; Tao BIAN ; Li ZHAO ; Li RUAN
Chinese Journal of Experimental and Clinical Virology 2006;20(2):33-37
BACKGROUNDMany epidemiological and experimental evidences prove that cervical cancers are strongly associated with genital high-risk types of human papillomavirus (HPV). HPV16 is present in 50% of the tumor specimens. Thus, it is important to develop vaccines against HPV16 and cervical cancer. The authors studied the expression of the HPV16 L1DeltaCE7N fusion protein in E. coli and observed its immunogenicity.
METHODSThe fragment of HPV16 L1DeltaC gene and the E7N gene were amplified by PCR separately; the fusion gene named L1DeltaCE7N was generated by fusing E7N to the C terminal of L1DeltaC then the chimeric gene was cloned into prokaryotic expression vector pGEX-2T and expressed in E. coli strain JM109. The L1DeltaCE7N protein expressed were detected by Western blot. Finally its immunogenicity was characterized in immunized mice.
RESULTSIt was proved that the sequence and open reading frame of fusion gene L1DeltaE7N was correct by sequencing; SDA-PAGE gel analysis showed that HPV16 L1/E7 fusion protein was highly expressed in E. coli; the protein was expressed as soluble form and the molecular weight was about 85 x 10(3). The fusion protein could be purified by affinity chromatography and gel filtration. The ELISA result indicated that L1/E7 could elicit specific antibodies against L1 and E7 in immunized mice. In vivo tumor protection test indicated that tumor formation was retarded or prevented in the mice after vaccination with L1/E7, when C57 BL/6 mice were challenged by syngeneic HVP16E6 and E7 transformed tumor cells.
CONCLUSIONHPV16L1/E7 fusion protein was expressed in E. coli, it can be a candidate for prophylactic and therapeutic vaccine for HPV16-associated infection and tumors.
Animals ; Blotting, Western ; Cell Line, Tumor ; Escherichia coli ; genetics ; Female ; Humans ; Immunization ; methods ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Neoplasms, Experimental ; immunology ; pathology ; prevention & control ; Oncogene Proteins, Fusion ; genetics ; immunology ; metabolism ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Papillomaviridae ; genetics ; immunology ; metabolism ; Papillomavirus Infections ; immunology ; pathology ; prevention & control ; Papillomavirus Vaccines ; administration & dosage ; immunology ; Recombinant Fusion Proteins ; immunology ; metabolism ; ultrastructure
4.Screening of traditional Chinese remedies for SARS treatment.
Zhi-min WANG ; Xiao-xin ZHU ; Xiao-lan CUI ; Ai-hua LIANG ; Guan-hua DU ; Jin-xiu RUAN ; Jian-ning SUN ; Peng-tao LI ; Shuo-ren WANG
China Journal of Chinese Materia Medica 2003;28(6):484-487
The SARS epidemic is breaking out worldwide. To select suitable herbal drugs for clinical uses is important and urgent amongst the controversial treatment proposals. Nine pharmacological experimental models were used to evaluate the comprehensive efficacy of traditional Chinese remedies by cross validation in different institutes. Eight drugs were optimized for controlling different symptoms of SARS.
Animals
;
Drug Evaluation, Preclinical
;
Drugs, Chinese Herbal
;
isolation & purification
;
therapeutic use
;
Humans
;
Multiple Organ Failure
;
prevention & control
;
Phytotherapy
;
Plants, Medicinal
;
chemistry
;
Severe Acute Respiratory Syndrome
;
drug therapy
5.Prediction and verification of biological basis and mechanism for traditional Chinese drugs of reinforcing kidney for supplementing essence in treating diseases related to deficiency of kidney essence
Chao WU ; Jia-hui WEI ; Han CHEN ; Tao-ren RUAN ; Zhuo-heng LI ; Ji-fen ZHANG ; Xiao-yu XU
Acta Pharmaceutica Sinica 2020;55(3):463-472
"Kidney essence" is a profound concept in the theory of traditional Chinese medicine. But its biological basis is unknown until now, resulting in the therapeutic effects of traditional Chinese drugs on reinforcing kidney for supplementing essence hard to be evaluated. This study aimed, to explore the potential biological basis and mechanism of traditional Chinese drugs of reinforcing kidney for supplementing essence on diseases related to deficiency of kidney essence through network pharmacology analysis on the intersection of targets of drugs and diseases. The targets for ingredients in