| Rabbit hemorrhagic disease virus(RHDV)infection mainly causes an acute,severe,highly contagious and highly dead disease in rabbits.The virus may cause bleeding and congestion of multiple organs in rabbits is called rabbit hemorrhagic disease(RHD).In this experiment,the RHDV VP60 segment gene was expressed in prokaryotic expression.Based on recombinant protein,preliminary study on colloidal gold and quantum dot immunochromatographic test strips.1.Prokaryotic expression of the RHDV VP60 segment geneAccording to the Gen Bank RHDV gene sequence,the VP60 gene(901bp-1740bp)was amplified by RT-PCR.Linked to p MD19-T cloning vector,after sequencing and identification,digestion by restrictive enzymes,ligated to construct recombinant expression plasmid p Cold TF-VP60-2 and converted to E.coli BL21(DE3).Expanded culture of positive bacteria identified by PCR,enzyme digestion and sequencing.Induction expression using IPTG(1 mmol/L).Perform SDS-PAGE analysis,Western-blotting identification,and affinity chromatography to purify protein ELISA.The results showed that the recombinant plasmid was successfully constructed and the expressed recombinant protein was 82.8 k Da.The optimum conditions for inducing expression were 37 ?,0.2 mmol/L and 6 h.5.16 mg/m L purified recombinant protein can be obtained by affinity chromatography.Indirect ELISA results of recombinant protein showed that the expressed recombinant protein can specifically bind to rabbit anti-RHDV antibody and has good reactivity.2.RHDV antibody detection of colloidal gold immunochromatographic test strips trial manufacturePreparation of rabbit anti-recombinant protein antibody.Colloidal gold-labeled recombinant protein is gold standard pad,rabbit anti-recombinant protein polyclonal antibody label C-line,goat anti-rabbit Ig G label T-line,design RHDV antibody test strip.Optimization of antibody dosage by T-lineand C-lines and specificity and sensitivity tests.Initial establishment of immunocolloidal gold test strips for RHDV antibody detection.5 serum samples of experimental immunization,5 serum samples of non-immune antibody negative and 154 serum samples of clinical immunization were tested.The results show that the developed colloidal gold test strip has good sensitivity and specificity,and its immune serum sensitivity can reach 1:16.Specific detection showed red bands on T-line in rabbit anti-RHDV serum.There were no color bands on the T line in serum of Clostridium perfringens type A and rabbits such as Pasteurella bacillus.All 5 positive samples were positive and 5 non-immunized sera were negative.154 clinical samples were tested by designed RHDV antibody test strips.The results showed that 150 were positive and 4 were negative.Provide a useful immunoassay strip for RHDV antibody level monitoring.3.RHDV antibody detection of quantum dot immunochromatographic test strips trial manufactureQuantum dot-labeled recombinant protein is gold standard pad,rabbit anti-recombinant protein polyclonal antibody label C-line,goat anti-rabbit Ig G label T-line,designed RHDV antibody test strip.Optimization of antibody dosage by T and C lines,as well as specificity and sensitivity testing and sample detection applications and sample detection application.Initial establishment of quantum dot test strips for RHDV antibody detection.The results showed that quantum dot immunochromatographic test strip has good sensitivity and specificity,and its immune serum sensitivity can reach 1:32.Specific detection showed red bands on T-line in rabbit anti-RHDV serum,while negative serum and rabbit Pasteurella bacillus serum showed no color bands on T-line.All 5 positive samples were positive,and 5 unimmunized sera were negative.154 clinical samples were tested by designed RHDV antibody test strips.The results showed that 150 were positive and 4 were negative.Compatible with the results of colloidal gold immunochromatographic test strips.It lays a foundation for the further development of quantum dot immunochromatographic test strips. |
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