OBJECTIVETo construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells.

METHODSHuman cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection.

RESULTSThe XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses.

CONCLUSIONSThis novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

Animals ; Baculoviridae ; genetics ; Cells, Cultured ; Cloning, Molecular ; Gene Expression ; Genes, Viral ; genetics ; Genetic Vectors ; Hemorrhagic Fever Virus, Crimean-Congo ; genetics ; Transfection ; Viral Core Proteins ; genetics

OBJECTIVETo explore the relationship between the structure and function at molecular level and the routes of transmission of Xinjiang hemorrhagic fever (XHF) virus.

METHODSS genes of five XHF virus strains were cloned, sequenced and compared with that of other Crimean-Congo hemorrhagic fever virus strains.

RESULTSIt was found that S genes of the five viruses had 1,672 nuclei tides, while ORF of them including 1,449 nuclei tides and coded with a protein of 482 amino acid. The nucleotides homology of Chinese isolates (93.0%-99.5%) was obviously higher than that of any other S genes strains identified in other countries'. Phylogenetic tree showed that all Chinese isolates clustered into one branch and could be further divided into another three groups.

CONCLUSIONThe sequential difference of S genes was not totally related to the host, areas and time of the viruses isolated.

Genes, Bacterial ; Genetic Variation ; Hemorrhagic Fever Virus, Crimean-Congo ; classification ; genetics ; Phylogeny

BACKGROUNDTo express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin.

METHODSThe human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E. coli. The gene expression was confirmed by Western blot assay. Rabbit was inoculated with purified antigen to stimulate the antibody generation. The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis. The separated cell clones were confirmed by immunofluorescence and Western blot assay.

RESULTSThe alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity. High titer antibody was generated. However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells.

CONCLUSIONSThe results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.

Animals ; Cercopithecus aethiops ; Cloning, Molecular ; Gene Expression ; Hantavirus ; Integrin beta3 ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Virus ; Vero Cells ; metabolism