Detection Of Classical Swine Fever Virus Antibody Based On E2 Protein Colloidal Gold And Quantum Dot Immunochromatographic Strip

Classical swine fever is a highly pathogenic infectious disease caused by the classical swine fever virus of the genus Flavivirus,which is harmful to pig farming,pig production and pig processing industry in many countries.In this study,detection of swine fever virus antibody based on E2 protein colloidal gold and quantum dot immunochromatographic strip.1.Prokaryotic expression of CSFV E2 geneIn this study,a recombinant plasmid pET-32a-E2 was constructed by using the sequence of an antigen epitope on E2 protein.The recombinant plasmid was transformed into E.coli BL21(DE3)for expression induction,solubility identification,optimization of induction conditions,purification of recombinant protein and identification of antigenicity.The results showed that the recombinant plasmid pET-32a-E2 was successfully constructed,and the recombinant E2 protein was induced.The recombinant protein was broken by ultrasound and existed as inclusion body.After purification and concentration,the expression of recombinant protein was 0.8 mg/mL.Western Bloting results showed that the recombinant protein was antigenic.Rabbit polyclonal antibody against recombinant E2 protein was successfully prepared by mixing recombinant protein with adjuvant.2.Establishment of Colloidal Gold Immunochromatography for Detection of Antibody to CSFVWe made colloidal gold solution and labeled recombinant protein with colloidal gold to optimize the labeling conditions.T line coated with Rabbit anti pig IgG and C lines was coated with rabbit polyclonal antibody against recombinant protein,and the concentration of T line and C line was optimized.An immunochromatographic strip was prepared to test the performance of the test strip.The results showed that the strip had good specificity and repeatability,and the standard positive antibody of CSFV could still be detected at 1:25 dilution,which proved that the strip had good sensitivity.The coincidence rate of immunochromatographic strip and CSFV antibody ELISA kit(IDEXX)was 92.1%.3.Establishment of a Quantum Dot Immunochromatographic Strip for Detecting Antibody to CSFVThe quantum dots were coupled with recombinant protein to optimize the conditions of coupling pH,activator and labelling quantity.The T line was coated with rabbit polyclonal antibody against pig IgG and C lines,and the concentration of T line and C line was optimized to prepare an immunochromatographic strip.The results showed that the strip had good specificity and repeatability,and the standard positive antibody of CSFV could still be detected at 1:30 dilution,which proved that the strip had good sensitivity.The coincidence rate of immunochromatographic strip and CSFV antibody ELISA kit(IDEXX)was 95%.