Pseudo Rabies Virus Strains Sl Gh, Gl Prokaryotic Expression Of Genes And Some Biological Characteristics Research

In this essay, clone, prokaryotic expression, preparing polyclonal antibodies, intracellular localization and some biological characteristics research of the Pseudorabies Virus(PRV) Strain SL gH gene and gL gene. The results of research such as follows:1. The bioinformatics analysis of gH gene The structure, antigenicity, motif and rare codon of gH gene and the encoding amino acid sequence were analysed by bioinformatics. The ORF of PRV gH gene is2061bp and encoding686amino acid residues, which is71.95KDa in theoretical molecular weight. The result of gH gene being analysed by bioinformatics showed that the G+C of this sequence was over76%, there was a CpG island between46bp to2004bp, and there were33rare codons. The result of gH gene amino acid sequence being analysed by bioinformatics showed that there was one signal peptide cleavage site between24and25amino acids, there was one transmembrane region in645-667amino acids, and there were26antigennic determinants. In addition, the result of subecellular localization demonstrates that the gH mainly located at cytoplasm.2. The expression plasmid construction, prokaryotic expression of gH gene and preparation of polyclonal antibody The one pair of primers was used to amplify gH gene by touchdown PCR. Then we began to T clone and constructed the expression plasmid pET-gH. Trough IPTG induction expression, gH gene could express well in Rosetta prokaryotic expression system. The optimized expression condition was0.4mmol/L IPTG at37℃5h, meanwhile the expression product existed as inclusion body. Western blot analysis proved that fusion proteins could react with anti-PRV polyclonal antibodies. The fusion protein was purified by inclusion body washing method. Then the purified protein was used to immunize the rabbits for preparing polyclonal antibodies. The antibodies titer was1:8.3. The expression plasmid construction, prokaryotic expression of gL gene and preparation of polyclonal antibody The one pair of primers was used to amplify gL gene by PCR. And then we began to T clone and constructed the expression plasmid pET-gL. Trough IPTG induction expression, gL gene could express well in Rosetta prokaryotic expression system. The optimized expression condition was0.6mmol/L IPTG at30℃5h, meanwhile the expression product existed as soluble protein. Western blot analysis proved that fusion proteins could react with anti-PRV polyclonal antibodies. The fusion protein was purified by Ni2+-Agarose His label protein purification kit. Then the purified protein was used to immunize the rabbits for preparing polyclonal antibodies. The antibodies titer was1:8.4. The subcellular localization of gH、gL gene products and polyclonal antibodies to the virus proliferation influence The infected PRV cells were detected by indirect immunofluorescence. The results indicate:The gH protein could be detected after PRV infected cells4h, located in the cytoplasm, with the viral replication gH protein expression increased, and which could be detected in syncretic cells12h. The gL protein could be detected after PRV infected cells4h, located in the cytoplasm, with the viral replication gL protein expression increased, and which could be detected in the cytoplasm membrane12h. One-step method was used to reserch polyclonal antibodies to the virus proliferation influence. The results indicate: The polyclonal antibody to the gH、 gL protein could influence PRV proliferation in cells, PRV virus titer lower compared to standard step growth curve, but which didn’t influence PRV spreading between cells.