The Study Of Construction Of Mutant F And Hn Gene Of Swine Newcastle Disease Virus And Cell Fusion In BHK-21Cell

In order to understand molecular mechanism of spread across the poison of infected pigs of the Newcastle disease virus JL01strain. First,we extracted the genome of bacteria from E.coil DE3(BL21), and design specific amplification primers of T7RNA polymerase (T7RNAP) gene,, PCR amplification was used to get T7RNAP genes from the genome of E.coil DE3(BL21) bacteria, and green fluorescent protein gene EGFP cloning to pSTK carrier of directional transfer vector of vaccinia virus by gene cloning and constructed the pSTK-T7-EGFP restructuring expression plasmid, transfected and expression the plasmid into BHK-21cell infected vaccinia virus of TK genetic mistakes strains, the fluorescence microscope observation records the fluorescence expression, results show that T7RNAP genes and EGFP gene won the expression on BHK-21cells. After that, PCR amplification methods was used to get F gene of Swine Newcastle disease virus, and got HN gene of Swine Newcastle disease virus by gene synthesis, gene cloning and recombinant PCR methods were used to constructed F and HN mutants and cloned into the pKS vector, pSTK-T7-EGFP expression plasmid of T7RNAP gene and expression plasmid of F and HN gene and F and HN mutants cloned into the pKS vector were co-transfected into BHK-21cells, observation of cell fusion by the Giemsa stain, the area and cell counting were used to detected the efficiency of cell fusion, SPSS statistical analysis, the results showed RF5of F gene mutant strain constructed of the attenuated strain Ireland as reference series, fusion rate of0.69+0.05, much higher than that of F gene and HN gene fusion control group (P<0.05), but RF3of waterfowl virulent strain NDV-Dove as a reference sequence of F gene mutant strain, fusion efficiency was0.25+0.02, significantly lower than the control group (P<0.05), therefore, there was a certain extent effection that the F protein cleavage site region of the gene sequences of Swine Newcastle disease virus to specific membrane fusion capacity, but the virulent strain F gene cleavage site mutants did not improve on cell membrane fusion Photosynthetic capacity, and attenuated strain F gene cleavage site mutants has bigger promotion to cell membrane fusion ability, this conclusion proved cell membrane fusion ability of Swine Newcastle disease virus F gene cleavage site and HN gene was higher than that of high virulent Newcastle disease virus. But the mutations of119and120amino acids poison(S-N)(RHN4group) of HN protein of swine Newcastle disease virus and F gene expression plasmid co-transfected into BHK-21cell, fusion results show that HN protein membrane fusion performance was improved to BHK-21cell. F genetic mutations and HN gene mutations strains were co-transfected into BHK-21cells, the fusion results show that effect was very obvious of waterfowl source virulent strain F protein schizolysis sites and HN protein double mutant of119and120amino acids to cell membrane fusion of BHK-21, infection ability of virulent strain of waterfowl and Swine Newcastle disease virus co-infected into BHK-21cell were improved, and membrane fusion ability of the weak strain F protein schizolysis sites and HN protein double mutants of119and120amino acids to BHK-21cell were improved, show that the ability of infected BHK-21cells of weak strain of Newcastle disease virus and Swine Newcastle disease virus will further enhance.