title: Construction and expression of a eukaryotic expression vector containing a fusion gene of the Hantaan virus S gene and hsp70 gene.
abstract: Hantavirus (HV) infection leads to a kind of severe systematic syndrome, hemorrhagic fever with renal syndrome (HFRS). Heat shock proteins (HSPs) can be used as adjuvants assisting soluble antigens to produce specific targets which can be attacked by cytotoxic T lymphocytes. For further research on HFRS vaccine, this study aimed to express Hantaan virus nucleocapsid protein (HTNV NP)-HSP70 fusion protein in COS-7 cells. First, an HTNV S gene encoding NP was amplified by PCR with a mutated termination code and cloned into eukaryotic expression vector pCDNA3.1(+), into which the full-length hsp70 gene had already been inserted, to form the S-hsp70 fusion expression vector pCDNA3.1(+)/S-hsp70. Then this recombinant plasmid was transfected into COS-7 cells by liposome, and eukaryotic expression of NP-HSP70 fusion protein was detected by immunocytochemistry and western blot. The results show that the eukaryotic expression vector pCDNA3.1(+)/S-hsp70 was successfully constructed and the NP-HSP70 fusion protein was effectively expressed in COS-7 cells. This study demonstrates that the NP-HSP70 fusion protein was expressed effectively from the pCDNA3.1(+)/S-hsp70 vector in a eukaryotic system and thus provides a basis for using this plasmid as a new DNA vaccine against HV infection.
authors.affiliation: Department of Digestive Diseases, Wuhan General Hospital of Guangzhou Command PLA, Wuhan, 430070, Hubei province, People's Republic of China.
authors.forename: Juan
authors.initials: J
authors.lastname: Gao
authors.forename: Bicheng
authors.initials: B
authors.lastname: Zhang
authors.forename: Shoujing
authors.initials: S
authors.lastname: Yang
authors.forename: Kainan
authors.initials: K
authors.lastname: Li
authors.forename: Hualin
authors.initials: H
authors.lastname: Xu
authors.forename: Yimin
authors.initials: Y
authors.lastname: Xiong
authors.forename: Yiming
authors.initials: Y
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Weitian
authors.initials: W
authors.lastname: Xu
authors.forename: Yi
authors.initials: Y
authors.lastname: Cheng
authors.forename: Guorong
authors.initials: G
authors.lastname: Zheng
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics* / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology; D002522:Chlorocebus aethiops; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D036022:Capsid Proteins; D018840:HSP70 Heat-Shock Proteins; D019444:Vaccines, DNA; D014758:Viral Core Proteins; C111782:nucleocapsid protein, Hantaan virus