Prokaryotic Expression And Immunogenicity Of The B2L And F1L Truncated Fusion Protein Of OrfV

Orf is a contagious disease caused by the orf virus(OrfV)infecting goats or sheep.The disease is highly contagious and has a high incidence.Once a sheep farm becomes ill,it will become an epidemic trend,which will seriously endanger the breeding.The development of the sheep industry.OrfV is a member of Poxviridae and Parapoxvirus,and is a linear double-stranded DNA virus.The B2 L and F1 L proteins are the envelope proteins of the OrfV.They have strong antigenicity and good conservation.They can induce strong humoral and cellular immune responses in the animal body.Therefore,the B2 L and F1 L proteins have become a new type of research on the OrfV.The target protein of the vaccine.In this study,the best effective antigen fragments of the two genes B2 L and F1 L were intercepted and fused by SOEPCR technology.The B2L-F1 L fusion protein was induced to express by IPTG.The purified B2L-F1 L fusion protein was used as the coating antigen to establish An indirect ELISA method based on the B2 L and F1 L recombinant proteins of OrfV.At the same time,the purified B2L-F1 L fusion protein is immunized with lambs and its immune indicators are tested to evaluate the immune effect of the B2L-F1 L fusion protein.Research and development provide a new direction.1.Construction and bioinformatics analysis of the B2 L and F1 L truncated fusion Genes of OrfV.The purpose of this research is to construct the B2L-F1 L fusion gene of the OrfV and conduct bioinformatics analysis of the B2L-F1 L fusion gene sequence.Use DNAstar software to analyze the B2 L and F1 L gene sequences respectively,intercept the main antigen regions of the B2 L and F1 L gene sequences,design two pairs of primers,and use the overlap extension PCR(SOE-PCR)technology to extract the main antigen regions of the B2 L and F1 L genes Fusion and ligation with the PMD18-T vector to construct a recombinant plasmid PMD18-T-B2L-F1 L.After plasmid PCR,double enzyme digestion and sequencing are verified to be correct,the bioinformatics online software is used to signal the encoded protein.,Transmembrane structure,hydrophilicity,antigenic index,antigenic determinants,flexibility,glycosylation binding site,phosphorylation binding site and secondary structure and tertiary structure prediction.The results showed that the B2L-F1 L fusion gene was successfully constructed,the size was about 1080 bp,which was consistent with the expected target fragment size;it is predicted that the protein has a signal peptide,no transmembrane domain,and has good hydrophilicity,antigenic index and flexibility.There are 17 antigenic determinants,2 glycosylation and 28 phosphorylation binding sites.In the secondary structure,?-helix and random coils account for more.2.Prokaryotic expression of the truncated fusion protein of OrfV B2 L and F1 L and preparation of polyclonal antibody.The purpose of this experiment is to construct a recombinant plasmid of OrfV PET-32a-B2L-F1 L,express B2L-F1 L fusion protein in E.coli,and prepare rabbit anti-B2L-F1 L fusion protein polyclonal antibody.The B2L-F1 L fusion gene was connected to the prokaryotic expression vector PET-32 a.After plasmid PCR,double enzyme digestion and sequencing,the PET-32a-B2L-F1 L recombinant plasmid was obtained,which was transformed into E.coli BL21 competent cells and induced by IPTG Express the B2L-F1 L fusion protein,and optimize the conditions for inducing expression of the protein.After the obtained B2L-F1 L fusion protein is purified by a Ni column,it is identified by Western-Blot,and the purified B2L-F1 L fusion protein is immunized with two New Zealand White rabbits,preparation of rabbit anti-B2L-F1 L fusion protein polyclonal antibody.The results showed that the PET-32a-B2L-F1 L recombinant plasmid was successfully constructed.The optimal induction temperature for fusion protein expression was 37 ?,the final concentration of IPTG induction was 0.2 mmol/L,and the induction time was 5 h.The B2L-F1 L fusion protein was obtained.The size is about 39 KD;Western-Blot results show that the B2L-F1 L fusion protein reacts positively with the positive sera of OrfV;after the purified B2L-F1 L fusion protein is immunized with New Zealand white rabbits,rabbit anti-B2L-F1 L polyclonal antibodies are successfully obtained The titer of rabbit 1serum was 1:419600 and rabbit 2 serum titer was 1:204800 by ELISA method.Western-Blot analysis showed that the B2L-F1 L fusion protein can react with the prepared rabbit anti-polyclonal antibody.The results show that the B2L-F1 L fusion protein is immunogenic.3.Establishment of an indirect ELISA method based on the B2L-F1 L fusion protein of OrfV.The purpose of this experiment is to use purified B2L-F1 L recombinant protein as the coating antigen to establish an ELISA detection method based on the B2 L and F1 L fusion proteins of the OrfVUse.the Founder Titration method to determine the protein coating concentration and serum dilution factor,protein coating method,skimmed milk powder blocking time,primary antibody and enzyme-labeled secondary antibody incubation time,enzyme-labeled secondary antibody dilution factor,TMB color time,negative and positive threshold The determination of the value is optimized,and the established ELISA method is evaluated through sensitivity,specificity and repeatability tests.The results showed that the best coating concentration of the fusion protein was 0.25 ?g/m L,the best dilution ratio of the primary antibody was 1:200,the blocking time of 5% skimmed milk powder was 1 h,and the best incubation time of the primary antibody and enzyme-labeled secondary antibody was 1 h,the best dilution factor of the enzyme-labeled secondary antibody is1:8000,the best color development time of TMB is 15 min,and the critical value of negative and positive is determined to be 0.358,and the sensitivity can reach 1:512.It is similar to that of FMDV(type A,Type O),FMDV 3ABC recombinant protein,Mycoplasma ovis,PPRV,and Brucella positive sera were all negative;the coefficient of variation for intra-assay and inter-assay repeatability was less than 15%.The results show that the test is based on the B2 L and F1 L genes of the OrfV,and the established indirect ELISA method has good sensitivity,specificity and reproducibility,which provides a rapid,rapid,and reliable method for the rapid differential diagnosis and immune antibody monitoring of OrfV.Simple serological diagnosis method.4.Study on the immunogenicity of OrfV B2 L and F1 L truncated fusion protein to lamb.The purpose of this test is to evaluate the immunogenicity of the B2L-F1 L fusion protein to lambs.The B2L-F1 L fusion protein immunization group,the Orf attenuated live vaccine group and the normal saline group were set up respectively,and were immunized by multiple subcutaneous injections in the neck for 1 month.Lambs were immunized twice,and then ELISA method was used to detect the content of OrfV specific antibodies and IFN-?,IL-2,IL-4,IL-6 cytokines in the serum of lambs immunized at different time periods.The results showed that both the attenuated aphtha vaccine and the B2L-F1 L fusion protein can stimulate the lamb body to produce OrfV specific antibodies.The antibody level of the B2L-F1 L fusion protein immunization group continued to increase after the second week of booster immunization,and in the fourth week Slightly lower,and very significant(P<0.01)higher than the attenuated live vaccine immunization group and normal saline group in 1 to 4 weeks.After the initial immunization,the OrfV specific antibody of the Orf attenuated live vaccine group maintained for 1 to 4 weeks.At the same level,and extremely significantly(P<0.01)higher than the normal saline group;Both the Orf live attenuated aphtha vaccine and B2L-F1 L fusion protein can stimulate the lamb body to secrete IL-2,but the content is low.There is no significant difference from the normal saline group in the first week.At the second and third weeks,the live attenuated aphtha vaccine is immunized.The group was significantly(P<0.05)higher than the B2L-F1 L fusion protein immunization group and the saline group,extremely significantly(P<0.01)higher than the saline group at the 4th week,and significantly(P<0.05)higher than the B2L-F1 L fusion group Protein immune group;After the B2L-F1 L fusion protein immunization group was boosted in the second week,the level of IL-2 secretion continued to increase in the third and fourth weeks,which was significantly(P<0.05)higher than the normal saline group;Both the aphthous live vaccine and B2L-F1 L fusion protein can stimulate the lamb body to secrete IFN-?,and the IFN-? content is extremely significant(P<0.01)higher than that of the normal saline group at 1 to 4 weeks.Compared with the B2L-F1 L fusion protein immunization group,the IFN-? content of the sheep aphthous attenuated live vaccine group was significantly higher in the first 1,2,and 4 weeks(P<0.05)than the B2L-F1 L fusion protein group,the third week no obviously different;Both the sheep aphthous attenuated live vaccine and the B2L-F1 L fusion protein can stimulate the lamb's body to secrete IL-4,and the amount of IL-4 secreted during 1 to 4 weeks was significantly higher(P<0.05)than the normal saline group,while the attenuated live vaccine group IL-4 began to secrete in the second week after the initial immunization,and it was significantly(P<0.05)higher than that in the saline group at 2 to 4 weeks.The B2L-F1 L fusion protein group was compared with the attenuated live vaccine group,and the B2L-F1 L fusion protein The group was significantly higher in the first week(P<0.05)than the attenuated live vaccine group,and the difference was not significant in the second to fourth weeks;Both the attenuated live aphtha vaccine and the B2L-F1 L fusion protein can stimulate the lamb's body to secrete IL-6,which was significantly higher(P<0.05)than the normal saline group at the second week,but at the third week the live attenuated aphtha vaccine group,B2 L The difference between-F1 L fusion protein group and normal saline group was not significant.In the 4th week,the B2L-F1 L fusion protein group was significantly(P<0.05)higher than the normal saline group,but there was no significant difference between the sheep aphthous attenuated live vaccine group and the normal saline group.The results show that the B2L-F1 L fusion protein can stimulate the humoral immune response and cellular immune response in lambs.