| Riemerlla anatipstifer,abbreviated as RA.It is an important bacterial pathogen that can widely cause Riemerella anatipestifer disease in poultry in a wide range,usually causing the disease of ducks,geese and turkeys.Significant economic losses,seriously restricting the healthy development of the poultry industry.Vaccine immune antibody evaluation and early diagnosis of Riemerella anatipestifer are more effective measures to control the disease.However,in production practice,due to the large number of RA serotypes and the lack of cross-protection of each serotype,the current market ELISA Antibody kits are few and of varying quality,limiting the development and application of RA antibody kits.Therefore,it is particularly important to establish an ELISA method that can both assess the level of RA immune antibodies and make a diagnosis of RA.In order to establish a novel indirect ELISA method for antibody detection of Riemerella anatipestifer,this study took the RA Guizhou isolate as the research object.By designing primers and amplifying the nucleic acid sequence of RA microporin porin gene,a recombinant expression plasmid p ET-32a-porin was constructed,transformed into E.coli BL21(DE3)and induced to express recombinant porin protein by adding IPTG,using Beyo Gold(?)His-tag Purification Resi purifies the recombinant protein,uses the purified Porin protein as an antigen to immunize experimental animals to prepare polyclonal antibodies,Western-Blot verifies the reactivity of the Porin protein,and finally uses the recombinant Porin protein as a coating antigen to establish a A novel indirect ELISA method suitable for the detection of Riemerella anatipestifer antibodies,aiming to provide technical support for the effective prevention and control of RA.1 Cloning and bioinformatics analysis of the porin gene of Riemerella anatipestiferIn order to study the molecular structure of porin gene of RA Guizhou isolate,the RA Guizhou isolate was taken as the research object.According to the published porin gene sequence of RA(CP002346.1),a pair of primers was designed to amplify the nucleic acid sequence of microporin porin gene of two serotypes(serotype 2,serotype 11)of RA,using DNAstar bioinformatics analysis software Homology comparison was carried out with the porin genes of 10 strains of RA included in Gen Bank and the porin genes of 8 serotypes of RA studied by domestic scholars.Predictive analysis of signal peptide,secondary structure,hydrophilicity,transmembrane region,subcellular localization,antigenic epitope and tertiary structure.The results showed that:the test successfully amplified the porin gene of RA Guizhou isolate with a size of 1 254 bp;the porin gene of the two serotypes of RA had a homology of up to 100%with the selected RA strain;The molecular mass is 47.68 k Da,contains 417 amino acids,the isoelectric point is 9.494,the 1-18 amino acids are the Porin protein signal peptide region(Sec/SPI)and the formation probability is 0.8111,and the secondary structure contains Alpha helix 18.47%(77/SPI)417),Extended strand 29.98%(125/417),Beta turn 5.52%(23/417),Random coil 46.04%(192/417).It has strong hydrophilicity and has a transmembrane helical region.The results of subcellular localization show that it is a membrane protein member with multiple potential epitope sites.Its dominant epitope region mainly includes 17-22,37-42,76-79,84-91,106-109,128-131,156-165,196-201,214-223,241-243,250-252,268-272,274-280,282-288,311-317,319-322,356-361,366-37-9 tertiary structural homology prediction 4,2-9The protein has a tubular structure,which is mainly composed ofα-helix,β-sheet,a large number of extension lines and random coils.The results showed that:RA Guizhou isolates have porin gene;porin gene is shared by many serotypes of RA and is highly conserved;Porin protein has multiple dominant epitopes,which can lay a solid foundation for the subsequent immunogenicity research of this protein.theoretical basis.2 Prokaryotic expression of Riemerella anatipestifer Porin protein and preparation of polyclonal antibodyTo express recombinant Porin protein and prepare its polyclonal antibody.The digested porin gene was connected to the prokaryotic expression vector p ET-32a,and the recombinant expression plasmid p ET-32a-porin was constructed after plasmid PCR,double digestion and sequencing identification,and p ET-32a-porin was transformed into E.coli BL21(DE3)and IPTG were added to induce expression,and the recombinant Porin protein was obtained by SDS-PAGE gel electrophoresis analysis.Combined with SDS-PAGE gel electrophoresis,the protein expression conditions were optimized,the expressed recombinant protein was purified,and the reactogenicity was analyzed by Western-Blot.The purified Porin protein was used as the antigen to immunize two clean-grade New Zealand white rabbits to prepare of anti-porin polyclonal antibodies.The titer of the polyclonal antibody was determined by ELISA,and its reactogenicity was analyzed by Western-Blot.The results showed that the constructed p ET-32a-porin was correctly identified by plasmid PCR,double enzyme digestion and sequencing;the size of the induced expression Porin protein was 63 k Da,and the optimal inducible expression conditions were:0.2 mmol/L IPTG at 30℃to induce expression for 3 h;Western-Blot results showed that the purified protein could react specifically with duck RA-positive serum and Porin polyclonal antibody,the titer of polyclonal antibody is as high as1:10~6 or more.The results showed that:p ET-32a-porin was successfully constructed;63 k Da recombinant Porin protein was induced to express;the protein had good antigenicity,which laid the material foundation for the establishment of RA antibody detection ELISA method.3 Establishment and clinical application of an indirect ELISA method for the detection of Porin protein of Riemerella anatipestiferAn ELISA method(Porin-ELISA)was established with recombinant Porin protein as coating antigen for antibody detection of RA.Using recombinant Porin protein as coating antigen,optimize ELISA reaction conditions(coating concentration,primary(second)antibody dilution,coating conditions,blocking solution selection,blocking time,primary(second)antibody incubation time and TMB color development time,etc.),according to 58 negative serum test results to determine the cut-off critical value of negative and positive,and comprehensively evaluate the established method through specificity,sensitivity,repeatability and shelf life tests,using Porin-ELISA and commercial ELISA antibodies The kit simultaneously detected 73 duck sera to compare the coincidence rate,and used Porin-ELISA method to detect antibodies in 801 duck sera in some areas of Guizhou Province(626 of which were immunized duck infectious serositis bivalent inactivated vaccine(serotype 1+serotype 2),and 175 duck sera were not vaccinated against duck infectious serositis vaccine).The results showed that the optimal protein coating concentration was 0.478μg/m L,the dilution ratio of primary antibody was 1:100,the coating conditions were 37℃for 1 h and then overnight coating at 4℃,the blocking solution was 1%BSA,and the blocking conditions were Blocking at 37℃for 2 h,incubation time of primary and secondary antibodies for 1 h,dilution of secondary antibody was 1:8 000,and TMB color development time was 10 min;the cut-off critical value of negative and positive was 0.458;this method was similar to AIV H5,H7subtype,NDV,Du CV,DTMUV and E.coli positive serum reaction results were negative;this method can detect RA positive serum,the sensitivity can reach 1:6 400 times or more;each serum The coefficient of variation of intra-assay and inter-assay detection was less than 10%;RA negative and positive serum were detected by enzyme-labeled strips stored at 4℃for 0-5months.0.458,the P/N value of the sample detected by the enzyme labeling strip stored for 5months was 4.830,which was still greater than 2.1;compared with the detection of commercial ELISA antibody kits,the coincidence rate of the two was 97.26%(71/73);The positive rate was90.10%(564/626),and the detection rate of infection antibody was 20.57%(36/175).The results showed that an antibody detection ELISA method(Porin-ELISA)for RA was successfully established;Porin-ELISA had good specificity,sensitivity and repeatability,and could still be stored at 4℃until the fifth month.Negative and positive sera can be detected well;compared with commercial kits,the two have a high coincidence rate;Porin-ELISA can be used for the detection of immune antibodies and infection antibodies. |
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