Construct And Express The Recombinant Expression Vectors For G Gene And G Gene Deletted Mutants Of Rabies Virus ERA Strain

According to the Genbank open sequence, nine pairs of primer were designed in order to amplify G gene and eight partial G genes: G1, G2, G3, G4, G5, G6, G7, G8 of rabies virus ERA strain, respectively. The G gene and eight partial G genes were obtained by reverse transcriptase polymerase chain reaction (RT-PCR), subsequently cloned into PMD18-T vector . Recombinant plasmids PMD-G1, PMD-G3, PMD-G5,PMD-G7 were digested by restriction endonuclease EcoR I and BamH I. And the PMD-G2, PMD-G4, PMD-G6, PMD-G8 were digested by BamH I and Pst I. Recombinant plasmids PMD-G and baculovirus transfer vector PAcSG2 were digested by EcoR I and Pst I. Then G gene and PAcSG2, G1, G2 and PAcSG2, G3> G4 and PAcSG2, G5, G6 and PAcSG2, G7, G8 and PAcSG2 were conjoined by T4 DNA ligase respectively. As aresult, we obtain the recombinant baculovirus transfer vector PAc-G and four deletion mutants PAc-GlG2, PAc-G3G4, PAc-G5G6, PAc-G7G8. Each mutant was deleted G residues range from 21 to 437 amino acid.We designed another pair of primer to amplify G gene and four partial G genes : G1G2, G3G4, G5G6, and G7G8 with the recombinant baculovirus transfer vectors as a model chain by PCR, then cloned into PMD18-T vector, and sucloned into eukaryotic expression plasmid vector pIRESneo. So we constructed the recombinant plasmids p-G, p-G1G2, P-G3G4, p-G5G6, p-G7G8.BHK-21 cells were transferred with the recombinant vectors by liposome-mediated gene transfer method. We obtain the positive cells clone after selection by geneticin G418. The aim DNA sequence can be amplified which demonstrated that the chromatin of BHK-21 cells encompassing the deletion G gene. And it may be helpful in studying the pathogenicity of G gene and finding out the amino acid domain about the cells fusion.