RESUMO
A cDNA containing the complete open reading frame of the M genome segment of Hantavirus R22 strain isolated from Rattus norvegicus in China, was amplified by polymerase chain reaction (PCR), and then cloned. The M segment is 3656 nucleotides in length with a predicted region of 3402 bases encoding a precursor glycoprotein of 1134 amino acids subsequently processed into viral glycoproteins 1 and 2 (G1 and G2). A strain comparison between R22 and SR11 (isolated from a rat in Japan), and Hantaan 76-118 (isolated from Apodemus in Korea), and Hallnas B1 (isolated from a bank vole in Sweden) revealed 95%, 74%, and 53% homologies at the deduced amino acid sequence level respectively. This suggests that the rodent host species may be a more important determinant of genetic relationships than geographic proximity. Six potential asparagine linked glycosylation sites (five in G1 and one in G2) were identified, and among them all are conserved in SR11, five in Hantaan virus and four in Hallnas B1 virus. Although different degrees of homology exist among these four viruses at amino acid sequence level, more than 90% of the cysteine residues are conserved, suggesting that structural homology may be very strong between the Hantaviruses. Genetic differences in the M segment genome of R22 and SR11 viruses, within the same serotype viruses, were found as random coding changes; some limited to single amino acids, others in clusters. A recombinant vaccinia virus that contained the fully activated M segment cDNA of R22 was constructed. This recombinant virus expressed two glycoproteins G1 and G2 identical to R22 virus G1 and G2 in molecular weight, cleavage pattern and cellular immunofluorescent patterns.
Assuntos
Glicoproteínas/genética , Orthohantavírus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , China , Clonagem Molecular , DNA Viral , Genes Virais , Vetores Genéticos , Orthohantavírus/isolamento & purificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ensaio de Radioimunoprecipitação , Ratos , Vaccinia virus/genéticaRESUMO
By inserting the LacZ gene into the plasmid containing ETEC LT-B antigen gene we obtained a recombinant plasmid which could express the beta-galactosidase and LT-B antigen, but has the Tc resistant gene. The K88ac antigen gene was then inserted into the BamHI site of the Tc resistant region in the above plasmid. The final recombinant plasmid has no antibiotic resistance gene but can stably express both LT-B and K88ac antigens, as well as the beta-galactosidase as a tracking marker.
