Research On The Different Eukaryotic Expression Of Non-structural Protein NS1?NS2A And NS1-2A Of Virulent And Avirulent Strains Of Japanese Encephalitis Virus

In this research,According to RNA isolation and RT-PCR amplified NS1? NS2 A and NS1-NS2 A gene fragment of JEV GZ strain and SA14-14-2 strain.And the purified products subcloned into pMD19-T Simple vector and pcDNA3.1(+)vector,Through bioinformatics analysis,the expression of eukaryotic expression plasmid and animal experimental to study the different eukaryotic expression of non-structural protein NS1?NS2A and NS1-2A from Japanese encephalitis virus Guizhou strain and SA14-14-2 strain.1.The clone of non-structural protein NS1 ? NS2 A and NS1-2A gene from Japanese encephalitis virus virulent and avirulent strainsSix pairs specific primers were designed and synthesized by Primer5.0 software according to DNA swquence of non-structural protein NS1?NS2A and NS1-2A gene from Japanese encephalitis virus GZ virulent strain(KC915016),SA14 virulent strain(U14163)and SA14-14-2 avirulent strain(AF315119)were published in GenBank,Cell cultures of total RNA of Japanese encephalitis virus as template were amplified NS1,NS2 A and NS1-2 genes by RT-PCR.Subsequently,the PCR products were cloned into pMD19-T Simple Vetor,and transformed into Top10 competent cells.Then plasmid were extracted after positive colones were idenified by colony PCR,after double enzyme digestion and sequence analysis to construct,the plasmids were named pMD19-T Simple-NS1 r,pMD19-T Simple-NS1 q,pMD19-T Simple-NS2 Ar,pMD19-T Simple-NS2 Aq,pMD19-T Simple-NS1-2Ar and pMD19-TSimple-NS1-2Aq.Results show that the difference is very apparent of virulent and avirulent strains' CPE,after them infected BHK-21 cells,the virulent strain infected cells produce obvious phenomenon of CPE was earlier than avirulent strain,and CPE phenomenon also has the typical differences,the virulent strain of plaque can be up to 2-3mm,avieulent strain produced smaller plaque only 0.5-1mm;And the cloned plasmids were sucessfully constructed.2.Construction of gene eukaryotic expression plasmid and bioinformatics analysis of non-structural protein NS1?NS2A and NS1-2A gene from Japanese encephalitis virus virulent and avirulent strainsBased on cloned plasmids,Double digested the T vector and recovered objective fragment,as the action of T4 ligase connection,linked the objective fragment and pcDNA3.1(+)vector and transformed into Top10 competent cells.Then plasmid were extracted after positive colones were idenified by colony PCR,after double enzyme digestion and sequence analysis to construct,the plasmids were named pcDNA3.1(+)-NS1 r,pcDNA3.1(+)-NS1 q,pcDNA3.1(+)-NS2 Ar,pcDNA3.1(+)-NS2 Aq,pcDN A3.1(+)-NS1-2Ar and pcDNA3.1(+)-NS1-2Aq.Through the MegAlign software to analysis the differences in gene sequences and amino acid,application software ExPASY(Protparam)program and EditSeq to analysis physical and chemical properties of the amino acid sequence of target protein,protein hydrophobicity analysis of ProtScale program at the same time,using DNAstar software Protan program for the purpose of protein secondary structure prediction,application the SIB bioinformatics resources portal ExPASY online across the membrane of the prediction,application TMpred program to analysisof proteins across the membrane.Results show that the eukaryotic expression plasmids were sucessfully constructed and through related software preliminary analysis of the change of the amino acids on the protein structure.Preliminary analysis found that virulent and avirulent strains NS1 protein,NS2 A and NS1-2A have not a signal peptide,belongs to the intracellular expression protein,the amino acid change does not affect the signal peptide,but the avirulent strain NS2 A protein compared with virulent strain in 99-121 more than form a transmembrane region,for other protein structure domain changes have little impact3.Research of eukaryotic gene expression and differences of non-structural protein NS1?NS2A and NS1-2A gene from Japanese encephalitis virus virulent and avirulent strainsEukaryon expression plasmids were transfected into BHK-21 cell to express by Lipofectamine? 2000 reagent.Eukaryotic expression vector expression was identified by indirect immunofluorescent technique and examine mRNA by RT-PCR and West-blotting technique.An intense fluorescence was observed 36 h posttransfection in the BHK-21 cells,extracted total RNA from transfection cells and control cells,the RT-PCR test resultdemonstrated that examined NS1,NS2 A and NS1-2A characteristic banding separately in 24 h,indicated that the recombinant plasmids can express corresponding mRNA in the BHK-21 cell.After 24 h,analysis by SDS-PAGE electrophoresis of the induced expressed protein and Western-blotting,specific fluorescent particles were detectedin cell by fluorescent antibody assay and got expressed protein that in line with the expected size about 46.8kDa?17kDa and64.4kDa.Results show that recombinant plasmids were expressed in BHK-21 cells and expressed protain has a certain reactogenicity.Select 90 Balb/c mice for four to five weeks,randomly divided into nine groups,each group of 10,was divided into six group,and three in the control group(normal saline group,JEV vaccine group and empty vector group),injection according to the set of immune immune method and the program.Immune interval of 2 weeks at a time,in the one immunity before,two immunity before,three immunity before and 2 weeks after the three immunity,separation of serum,use of encephalitis by antibody ELISA detection kits to experimental animals humoral immunity levels.Three weeks after the acquisition three immunity animal anticoagulant use flow cytometry measure to test CD4+and CD8+T cell levels,detection of animal cell immunity levels.The results show that the constructed recombinant eukaryotic expression plasmids could not induce the immune mice produce higher levels of humoral immunity.Construct the recombinant eukaryotic expression plasmid of immune mice can significantly or very significant increased the content of CD4+cells;Content of CD8+cells also increased at the same time,it means that Build the eukaryotic expression plasmid induces the body's immune cells.Nonstructural protein recombinant eukaryotic plasmid of virulent and avirulent strains of cellular immunity is compared between the same group of immune effects of CD4+cells:the influence of the virulent and avirulent strains strain NS1 and NS1-2A immune group,the virulent strain immune group slightly higher than the avirulent strain immune group,the virulent and avirulent strains strain of NS1 immune group significant difference(P<0.05).The avirulent strain NS2 A immune group increased the content of CD4+cells was slightly higher than the virulent strain immune group.Compare between the same group of immune effects of CD8+cells:the influence of the virulent and avirulent strains strain NS1,NS2 A and NS1-2A immune group,the virulent strain immune group slightly higher than the avirulent strain immune group.Comprehensive analysis,according to the construction ofthe recombinant eukaryotic expression,eukaryotic expression plasmid can enhance cellular immunity in mice,significantly or extremely significant increase the content of CD4+cells,can increase content of CD8+cells,virulent poison group restructuring eukaryotic plasmid in the expression of cell immunity in mice is better than avirulent poison group.