新疆出血热病毒糖蛋白基因的克隆与表达

Cloning and Expression of Xinjiang Hemorrhagic Fever Virus Glycoprotein Gene

On the basis of sequencing and analyzing of the whole M genes(encoding viral membrane antigen glycoprotein) of three Crimean Congo hemorrhagic fever viruses(CCHFV),the Chinese isolates(Xinjiang hemorrhagic fever virus,XHFV),we first expressed the glycoprotein (GP) gene of the prototype human origin XHFV strain BA66019 in eukaryotic cells and investigated the expression profiles.Three eukaryotic expression plasmids were constructed starting from ATG 78 (the first start codon of the deduced entire XHFv GP precursor gene positioned between the 78 th ～80 th nucleotides),ATG 93 and ATG 3084 (the potential start codon for G1 precursor gene).The constructs were transfected to COS-7 cells and the expressed products were characterized as membrane-bound proteins which could induce cell fusion This was much more apparent for recombinant plasmid with ATG 3084 A recombinant baculovirus was further created harboring full length GP gene(starting from ATG 78 ) and the expression could also result in the membrane fusion as well as swelling of the infected Sf9 cells The insect cell expressed G1 was smaller in M W than natural G1 (approximately 67kD) on SDS-PAGE and no recombinant G2 band was detectable Western-blot only detected the native G1,while there was no specific corresponding band of recombinant G1 These data suggested that G1 was structurally and functionally important in XHFV and the glycosylation may have great influence on M W as well as antigenicity This study provides a foundation for the future study of viral pathogenesis ,antigenicity and immunity,that are the theoretical and experimental backgrounds for vaccine

On the basis of sequencing and analyzing of the whole M genes(encoding viral membrane antigen glycoprotein) of three Crimean Congo hemorrhagic fever viruses(CCHFV),the Chinese isolates(Xinjiang hemorrhagic fever virus,XHFV),we first expressed the glycoprotein (GP) gene of the prototype human origin XHFV strain BA66019 in eukaryotic cells and investigated the expression profiles.Three eukaryotic expression plasmids were constructed starting from ATG 78 (the first start codon of the deduced entire XHFv GP precursor gene positioned between the 78 th ～80 th nucleotides),ATG 93 and ATG 3084 (the potential start codon for G1 precursor gene).The constructs were transfected to COS-7 cells and the expressed products were characterized as membrane-bound proteins which could induce cell fusion This was much more apparent for recombinant plasmid with ATG 3084 A recombinant baculovirus was further created harboring full length GP gene(starting from ATG 78 ) and the expression could also result in the membrane fusion as well as swelling of the infected Sf9 cells The insect cell expressed G1 was smaller in M W than natural G1 (approximately 67kD) on SDS-PAGE and no recombinant G2 band was detectable Western-blot only detected the native G1,while there was no specific corresponding band of recombinant G1 These data suggested that G1 was structurally and functionally important in XHFV and the glycosylation may have great influence on M W as well as antigenicity This study provides a foundation for the future study of viral pathogenesis ,antigenicity and immunity,that are the theoretical and experimental backgrounds for vaccine development