The Construction Of PVAX1-S6 Containing GCRV-Ⅱ S6 Gene And Studies On Its Immune Efficacy

Grass Carp is the most important freshwater fish in China,and its output accounts for about 20% of the total freshwater fish culture in China.However,the hemorrhagic disease caused by Grass Carp reoviral(GCRV)has caused huge economic losses to Grass Carp breeding in China,seriously affecting the healthy development of Grass fish breeding industry.GCRV is our country the first aquatic reovirus to separate,and It is also the most virulent and harmful pathogen of Aquareovirus.GCRV genome is points 11 segments of double-stranded RNA virus,mutate rapidly and complicated etiology and lack of effective prevention and control measures is the main cause widespread,rapid and continuous.Vaccine immunization is the most effective measure to prevent and control the hemorrhagic disease of grass carp,among which inactivated vaccine and attenuated vaccine are the most widely used,which play a positive role in the prevention and control of grass carp hemorrhagic disease.However,the inactivated vaccine could not enter the main histocompatibility complex I(MHC I)type antigen presentation pathway,and could not effectively induce the cytotoxic T cell(CTL)reaction.The demand for antigens was large,the duration of immunity was short,and the immune effect was relatively unsatisfactory.The attenuated vaccine can lead to virulence reversion,which may cause disease.Therefore,it is urgent to develop a more efficient and safe new vaccine for grass carp haemorrhagic disease.DNA vaccine has high safety and long duration of immune protection,which can induce early immune response.Its mechanism is to synthesize antigenic proteins through the transcription system of host cells,thus inducing immune animals to produce immune responses to the target antigenic proteins,so as to prevent and treat diseases.The technique involves cloning an exogenous gene that encodes a known target antigenic protein onto a eukaryotic expression vector and then injecting recombinant plasmid DNA into animals to achieve eukaryotic expression.Compared with traditional vaccines,DNA vaccines have the advantages of polyvalent vaccine,good immune effect,low production cost,long protection period and good safety.Therefore,in this study on the current popular type Ⅱ GCRV(GCRV-Ⅱ)for the purpose of main structure of protein-coding genes S6 genes,we construct eukaryotic expression recombinant plasmid p VAX1-S6,then identificate the expression of the recombinant plasmid in vitro effect and the antigenicity.By fish experiment we can evaluate the immune effect of fish,and evaluate DNA vaccine feasibility based on GCRV-Ⅱ S6 gene,the main research content includes the following two parts:(1)construction of recombinant plasmids and in vitro expression identificationTaking GCRV-Ⅱ new epidemic strains Hu Nan1307 as the research object,its whole ORF S6 gene by polymerase chain reaction(PCR)amplification,cloning into the eukaryotic expression vector p VAX1,enzyme cut the validation and then extract p VAX1-S6 plasmid.The transfected eukaryotic FHM was transfected with liposomal transfection and expressed instantaneously.The protein expression of S6 gene was identified by Western blot.The results show that,the GCRV-Ⅱ S6 gene was successfully cloned into p VAX1 carrier,and we obtained p VAX1-S6 plasmid;The recombinant plasmid was successfully expressed in FHM cells and identified by Western blot,and the expressed protein size was about 70 k Da.(2)in vivo test of DNA vaccine(fish immunity test)In this study,grass carps were divided into four groups: PBS control group,p VAX1 empty vector group,weakened virus vaccine group,and nucleic acid vaccine p VAX1-s6 group.The virus was inoculated two weeks later.Changes in the relative expression of 6 immune-related genes within 12 weeks after immunization were detected by fluorescence quantitative method,and specific antibody changes in immune grass carps were detected by ELISA method to monitor the protective effect of immune grass carps on strong toxic attack.These indexes were used to evaluate the immune effect of the genetic engineering vaccine on grass carp.The results showed that,when the nucleic acid vaccine p VAX1-s6 was injected into grass carp,the grass carp developed antibodies and maintained high levels for a long time.The relative expression levels of six immune-related genes in grass carp were up-regulated and then decreased.The protection rate of recombinant DNA vaccine against grass carp was more than 80%.Conclusion:we built S6 gene recombinant plasmid p VAX1-S6 successfully and the effect of expression in vitro is good,the proteins expressed can be a very good identification of GCRV-Ⅱ antibody,showing good immunogenicity;p VAX1-S6 nucleic acid vaccine by intramuscular immunization grass carp can be induced stronger humoral immunity and cellular immune response,and immune of grass carp infected relative protection ratio of 80%,with strong p VAX1-S6 nucleic acid vaccine immune to GCRV-strong Ⅱ strain has good protection effect,is an ideal candidate vaccine.