Genetic Engineering Study On Immunogen Of Fish Neuronecrosis Virus Mucosal Vaccine

Viral nervous necrosis(VNN)can cause viral neural necrosis of fish,and is one of the most harmful viral diseases in aquaculture in China,especially in the southeast coastal areas of China.The pathogen of this infectious disease is Nervous necrosis virus(NNV),according to reports,more than 30 types of ma rine fish have been affected by VNN during the seedling stage and during the cultivation process,especially causing high mortality rates for grouper larvae an d juveniles,and causing severe economic losses to marine fish farming.The p urpose of this experiment is to study the preparation of a mucosal vaccine that can effectively preventand and treat viral neuronecrosis through mucosal immun ization(oral mucosal immunization and immersion mucosal immunization)of gr ouper,so as to break the limitations of traditional immunity.E.ictaluri is a G ram-negative facultative intracellular pathogen that can cause intestinal septicemi a in catfish.This disease was first found in dying catfish in Georgiaand Alaba ma in 1976.It can enter the fish body through the gastrointestinal tract,gills,skin and other mucosal tissues,or into the body of the mouth,nostril(nose),and cause spotted catfish intestinal sepsis.Studies have shown that the outer m embrane protein(OMP)N encoded by E.ictaluri is involved in adhesion and invasion,and conferred its virulence properties.The encoded omp N1 protein pa rticipates in the anchoring of the peptidoglycan layer between the inner and ou ter membranes of the flagella and participates in the transduction of protein sig nals,and is easily recognized by infected hosts,so we believe that the omp N1 protein has a transmembrane effect,Plays an important role in the transmucos al pathogenic mechanism of E.ictaluri.Crp and Ton B proteins are two other proteins encoded by E.ictaluri.Theyare involved in the pathogenesisof ESC a nd are important virulence factors of E.ictaluri.In summary,according to the transmucosal pathogenic mechanism of E.ictaluri and knock out its pathogeni c protein,the coat protein MCP of fish neuronecrosis virus,which does not ha vemucosal immunity,can beknocked into E.ictaluri The genome was expresse d and the corresponding mucosal vaccine was prepared.Objective:Fused with the outer membrane protein N1(omp N1)of E.icta luri and the capsid protein(MCP)of neuronecrosis virus,a gene recombinant f usion protein(MCP-omp N1)with transmucosal immunity was expressedand puri fied in engineering bacteria To lay the foundation for the preparation of a mucosal immune vaccine against viral viral necrosis of fish.Secondly,bacterial gen e editing technology was used to knock out the pathogenic genes Ton B and Cr p of E.ictaluri,and to knock in the necrosis virus capsid protein(MCP)gene into the lack ?Ton B attenuated E.ictaluri genome and wild type The genome of E.ictaluri and expressed,The obtained new strain can be used for preparin g a mucosal immunization vaccine for preventing necrosis virus and E.ictalur.Methods:1.The gene sequences of the coat protein MCP of fish neurone crosis virus and the outer membrane protein omp N1 of E.ictalurie were obtai ned from the NCBI Gen Bank library,and the sequence optimization and gene synthesis of the two were performed.Prokaryotic expression vector were constr ucted: MCP-omp N1 p ET28 a,MCP p ET28 a,and omp N1 p ET28 a.Then the fusi on proteins MCP-omp N1,MCP and omp N1 were respectively induced and expr essed in Escherichia coli,and the proteins were purified by inclusion body pur ification and dialysis renaturation.The purified MCP and omp N1 proteins were used as antigens to immunize the mice.The total serum Ig G level in the seru m was measured by ELISA and the specificity of the antiserum was detected by Western-blot.2.To knock out the pathogenic genes Crp and Ton B,construc t gene knockout recombinant vectors: Crp-sg RNA1-Donor SX Pcrispr,Crp-sg RN A3-Donor SX Pcrispr,Ton B-sg RNA-Donor SX Pcrispr,These recombinant plasmi ds were electrotransformed into competent cells of E.ictaluri respectively.Aft er double-resistance screening,PCR,and sequencing verification,the attenuated E.ictaluri strains that successfully knocked out Crp and Ton Bwere finally sele cted.3.Knock-in of foreign gene MCP,and construct gene knock-in recombina nt vectors: MCP-?Ton B p K18 mobsac B,MCP-p K18 mobsac B,These recombinan t plasmids were electrotransformed into Attenuated Edwardsiella spp.And wildtype Edwardsiella spp.And they were verified by Kana resistance,20%sucrose,PCR,and sequencing in order to finally obtain a strain that successfully knock ed in the foreign gene MCP.Results:1.SDS-PAGEresults showed that the pure fusion protein MCP-om p N1 was obtained by prokaryotic expression and purification.Further ELISA te st results showed that compared with the PBS group,the total serum Ig G level s in the mice of the MCP and omp N1 groups were significantly increased.We stern Blotting results also showed that the purified MCP-omp N1 fusion protein had MCP antigenicity and also omp N1 antigenicity.2.The results of gene knockout experiments showed that positive clones were screened by double resistan ce of kana and chloramphenicol after electrotransformation.extracting the geno me,PCR was performed,and the PCR products were identified by 1.5% agaro se gel electrophoresis.The target bands are smaller than the unedited negative control genes.Alignment of the sequence results showed that ?Crp1 was parti ally deleted and mutated,?Crp3 knocked out 80 bp at 372?452 bp comparedt o the original Crp gene,?Ton B knocked out 79 bp from 336 bp to 415 bp c ompared to the original Ton B gene.The above experimental results proved that the Crp and Ton B genes were successfully knocked out to obtain a virulent E.i ctaluri strain.3.Kana resistance and 20% sucrose screening positive monoclon als,After extracting its genome and performing PCR and agarose gel electroph oresis,it was found that the target bands were about 1 kd larger than the une dited negative control band.The sequence comparison results showed that the t arget bands were larger than the unedited negative control because the foreign gene MCP had successfully knocked into the genomes of the attenuated E.ictal uri' strain and wild-type E.ictaluri.Conclusion: In this experiment,we got the MCP-omp N1 protein fused wi h the fish neuronecrosis virus capsid protein MCP and E.ictaluri outer membra ne protein omp N1 by prokaryotic expression.The results can providea base for the study whether the fusion protein MCP-omp N1 can be used as amucosal va ccine against neuronecrosis virus.Crisprca9 successfully knocked outthe Crp an d Ton B genes of E.ictaluri,and obtained the ?Crp's attenuated E.ictaluri's st rain(Edwarsiella ictaluri str.Crp ?Crp)and ?Ton B's attenuated E.ictaluri's str ain(Edwarsiella ictaluri str.Ton B ?tonB),It laid the foundation for knocking in the MCP gene of the capsid protein of exogenous neuronecrosis virus and prep aring the immersion / oral vaccine for the anti-Edwardella aquaculture industry.The suicide plasmid knockout system successfully knocked in the necrosis virus capsid protein MCP gene,A new attenuated E.ictaluri's strain(Edwarsiella ict aluri str.TOM ton B::mcp)and an inactivated wild-type E.ictaluri's strain(Ed warsiella ictaluri str.MCP mcp)were obtained,respectively,and the strains ca n be used to prepare a mucosal immune vaccine for preventing necrosis virus and E. ictaluri.