| Japanese encephalitis(JE)is a zoonotic disease,which was caused by the Japanese encephalitis virus(JEV).The main manifestations of the disease are neurological symptoms and reproductive disorders of swines,causing great damages to human healthy and breeding industry.At present,according to the change of epidemic dominant strains,the expansion of epidemic range and distribution,the vaccine is still the most powerful guarantee for preventing this disease.In this study,JEV GZ strain and SA14-14-2 strain were adaptively cultured on cells,and subjected to mouse brain inoculation,after specific designed 6 pairs of primers to amplify and clone pr M/M,pr M/E and NS1-NS2 A genes,constructing eukaryotic expression vectors.Through bioinformatics analysis and immunization test of animal to study proteins pr M/M,pr M/E and NS1-NS2A(NS1-2A)difference in eukaryotic expression and immune effects.This will lay a foundation for further research on the function of proteins of JEV and further research on vaccines.The JEV GZ strain and SA14-14-2 strain were successively passaged on BHK-21 and Vero cells for 30 passages,and the cytopathic effect(CPE)and the 5th,10 th,15th,20 th,25th JEV strains were observed.The results showed that the number of cytopathic changes increased with the extension of time after JEV GZ and SA14-14-2 were infected different cells,the characteristics of Vero cells were similar to BHK-21 cells,but the cytopathic changes of BHK-21 cells were more obvious than Vero cells,which was more suitable for the cultivation of the virus,the difference is apparent of JEV GZ and SA14-14-2 strains`s CPE,which were infected BHK-21 and Vero cells.JEV GZ strain infected cells produce obvious phenomenon of CPE was later than SA14-14-2strain,and CPE phenomenon also had typical differences.After 15 generations of BHK-21 and Vero cells,the titer of JEV was kept in a relatively stable range,and the virus content was relatively stable;Through the determination of the virulence of JEV GZ and SA14-14-2 strains on suckling mouse,which the the virulence of GZ strain was more obvious than SA14-14-2 strain.According to the JEV GZ strain and SA14-14-2 strain,six pairs of specific primers weredesigned and synthesized.Cell cultures of total RNA of SA14-14-2 strain and GZ strain as template were amplified pr M/M?pr M/E and NS1-2A genes by RT-PCR,the expected sizes were501 bp?2001 bp and 1737 bp,then they were cloned into PMD19-T vector and transformed into DH5?competent cells,and subjected to bacterial PCR detection,double enzyme digestion and sequencing identification.The results showed that the cloning plasmids of pr M/M,pr M/E and NS1-2A genes of JEV SA14-14-2 and GZ strains were successfully constructed.The pr M/M?pr M/E and NS1-2A were cloned into eukaryotic vector pc DNA3.1(+),and the recombinant plasmids DNA were identified by enzyme digestion,and the result indicate the recombinant plasmids conformed to a reading frame.The recombinant plasmids pc DNA3.1-pr M/M-r ? pc DNA3.1-pr M/E-r ? pc DNA3.1-NS1-2A-r ? pc DNA3.1-pr M/M-q ?pc DNA3.1-pr M/E-q and pc DNA3.1-NS1-2A-q(r represents SA14-14-2 strain with weak virulence,q represents GZ strain with strong virulence)were constructed.To analysis the differences in gene sequences and amino acid,physical and chemical properties of the amino acid sequence of target protein,protein hydrophobicity,protein secondary structure prediction and protein transmembrane situation,through the software DNAstart(Meg Align),Ex PASY(Protparam),DNAstart(Edit Seq),Prot Scale,DNAstart(Protean),(http ://smart.embl-heidelberg.de/)website.Results show that the eukaryotic expression plasmids were sucessfully constructed and through related software preliminary analysis of the avirulent strain pr M/E protein compared with virulent strain in 267-278 more than form a low complex region,the change of the amino acids for other protein structure domain changes have little impact.Transfected the constructed recombinant expression plasmids into BHK-21 cell to express by Lipofectamine? 2000 reagent,the recombinant expression plasmids was identified by indirect immunofluorescent and Westean-blotting technique,it was found that the corresponding target bands could be detected by RT-PCR,Six eukaryotic expression plasmids in BHK-21 showed specific green fluorescence by indirect immunofluorescence technique,and about 19 k Da,72.4k Da and 64.4 k Da specific bands were detected by Western-bolting.The results showed that the eukaryotic expression plasmids could be expressed in BHK-21 cells and had immunological activity.It was found that six kinds of recombinant plasmids could induce humoral immunity in mice,and the immune effect of recombinant eukaryon in GZ strian group was slightly better than that in SA14-14-2 strain group,among which,after immunization for 4 weeks,the recombinantplasmid pc DNA3.1-pr M/M-r immunization group and pc DNA3.1-pr M/M-q immunization group had significant differences(P<0.05),after 6 weeks of immunization,the levels of antibodies secreted by mice immunized with pc DNA3.1-pr M/E-q were significantly higher than those immunized with pc DNA3.1-pr M/E-r(P<0.01),the pc DNA3.1-NS1-2A-r secreted higher antibodies than the pc DNA3.1-NS1-2A-q immunized mice(P>0.05).Through the mouse challenge protection experiment,the protection rate of recombinant plasmids pc DNA3.1-pr M/M-q,pc DNA3.1-pr M/M-r and pc DNA3.1-pr M/E-r were found to be 30%,pc DNA3.1-pr M/E-q and pc DNA3.1-NS1-2A-r were 40%,pc DNA3.1-NS1-2A-q was 50%and the protection rate of inactivated vaccine was 70%,it shows that the recombinant expression plasmid against the JEV GZ strain can produce a certain protective effect. |
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