| Dairy cow mastitis is one of the most critical diseases restricting the healthy development of dairy cattle breeding industry.It is a common disease in dairy cattle breeding industry.After the disease occurs,it not only affects the quality of milk,but also harms human health and increases breeding costs.In particular,the latent mastitis in which the symptoms are invisible to the naked eye occurs in dairy cows,which is often ignored by people for treatment,which causes greater economic losses.Therefore,how to make early diagnosis of dairy cow mastitis has attracted more and more attention.At present,the diagnosis technologies related to mastitis at home and abroad have certain defects,and most of them can only be qualitative,not quantitative detection,which is not conducive to the early diagnosis of mastitis.At present,serum amyloid A3(M-SAA3)in milk has become a highlighted research in the diagnosis of mastitis.In this study,a prokaryotic expression system was used to prepare the bovine M-SAA3 recombinant protein,and the obtained recombinant protein was used to immunize mice to prepare corresponding monoclonal antibodies(mAbs).Finally,the principle of double antibody sandwich method was used to establish a method based on time-resolved fluorescence immunochromatography.The product of quantitative detection of M-SAA3 can meet the needs of rapid detection beside cattle,in order to provide technical support for early diagnosis of mastitis in dairy cows.In this study,the bovine SAA3 gene sequence in GenBank was used as a template,and after codon optimization,primers were designed to amplify the M-SAA3 gene,and a 426bp target gene fragment was amplified.Connect the target gene to the pET-30a(+)vector,and transform E.coli Trans 10 competent cells for gene cloning.After correct sequencing,it was transformed into E.coli BL21(DE3)competent cells for prokaryotic expression.After induction by IPTG at 20℃ for 18h,SDS-PAGE and Western blotting were used to identify the expression of M-SAA3 gene.The results showed that the pET-30a(+)-M-SAA3 recombinant plasmid was successfully constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells,and the expression was induced by IPTG,and then identified by SDS-PAGE and Western blotting.The results showed that the M-SAA3 gene was successfully expressed in E.coli and the target protein was about 15kDa,existed in the form of inclusion bodies.Purified the expressed protein by a nickel column,a high-purity target protein was obtained.The recombinant M-SAA3 protein purified by a nickel column was used as the immunogen,and BALB/c mice were immunized with multiple subcutaneous injections on the back to prepare anti-M-SAA3 mAbs.After 4 times immunizations,the mouse spleen cells with higher immune titers which screened out by indirect ELISA was uesd for fusion with SP2/0 cells.After multiple rounds of subclonal screening,7 strains which could stable secretoiy anti-M-SAA3 mAbs were obtained.Then indirect ELISA and Western blotting experiments were carried out to analyze the titer,subtype and specificity of mAbs.The results showed that the titer of these 7 cell lines were ranged from 1600 to 204800,all of which belonged to the IgG3 subclass.Western blotting results showed that the prepared monoclonal antibody 3B2B4 can specifically recognize the natural M-SAA3 protein.In summary,this study successfully prepared the bovine M-SAA3 recombinant protein and the monoclonal antibody.Based on the principle of double antibody sandwich method,self-made M-SAA3 antibody and purchased SAA antibody were used to develop fluorescent quantitative immunochromatographic detection products.After antibody raw material screening,process optimization of fluorescent microsphere labeling antibody,and fluorescence immunochromatographic detection test strip process optimization,the standard curve was made and the performance of blank of limit,recovery,linearity,precision,stability and sample test of the kit were evaluated.The results showed that Medix SAA-2 mAb coating and 3B2B4 mAb labeling were the best pair.The mass feed ratio of fluorescent microspheres and antibody was 40:1,the molar ratio of coupling agent to fluorescent microspheres carboxyl group was 2:1,the cross-linking buffer was 50mM MES pH6.0,which was the best process for labeling antibodies with fluorescent microspheres.The working concentration of the fluorescent microsphere-labeled antibody was VC:VT:Vs=1:7:6,the concentration of the coated antibody on the NC membrane was 1.0mg/mL,the sample volume was 100 μL,and the reaction time was 5min,which were the best conditions of fluorescence immunochromatographic detection strip.The four-parameter fitting curve equation of the standard curve of the kit was y=(1.21601-0.00481)/[1+(x/14.61922)^(-0.78088)]+0.00481,and the linear correlation coefficient was R2=0.9991.The blank limit of the kit is 0.079 mg/L,the recovery rate was in the range of 85%-115%,the linearity test result was R>0.99.the precision test result within the batch and the coefficient of variation were both less than 15%.Thermal stability test results showed that the relative decrease of the fluorescence T/C value of the sealed kit when stored at 37℃ for 14 days was<15%,and the correlation coefficient of the sample comparison test between the self-made kit and the Shanghai Blue-gene kit was 0.9762.In summary,the kit developed in this experiment has the advantages of simple operation,high sensitivity,and low cost.It can be used as a fast and accurate detection method for the detection of bovine SAA,and can meet the needs of clinical determination. |
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