Prokaryotic Experession Of VP2 Protein Of Porcine Bocavirus And Establishment And Application Of Indirect-ELISA Method

Porcine Bocavirus(PBoV),since be firstly found by the Swedish scientists in 2009,has began to be widely reported in the worldwide.In order to effectively detect PBoV,to develop the relevant detection method is imperative.In this study,the indirect ELISA of PBoV was established.This study selected a length of conservative genetic region with better antigenicity in PBoV VP2 gene for prokaryotic expression.The target gene was extracted from PBoV positive samples and the accuracy of the target gene was verified by sequencing.With the construction of recombinant plasmid pET-32a-PBoV-VP2,it was introduced into BL21(DE3)expressing strain.And At 37 degree centigrade,IPTG inducer was added to the inducible expression 5 h with concentration of 1mM.Six histidine-tagged recombinant proteins were expressed and the size was about 41 kD.The recombinant protein was identified as inclusion body,and the recombinant protein was purified and renatured.The results showed that the recombinant protein had a good specific reaction with PBoV positive sera and could be used as a specific antigen for the construction of indirect ELISA method.The recombinant protein after purified and renatured was used as coating antigen for indirect ELISA.The concentration was 8.25 ?g/mL;the primary anti-dilution ratio was 1:100;the secondary anti-dilution ratio was 1: 4000,confining liquid was 2% gelatin;the sealing time was 2 hours and the developing time was 15 min.The establishment of the indirect ELISA for PBoV was under these conditions.After the specificity test,the serum threshold test and within-between repeated test,the results showed that the indirect ELISA was with a good specificity,and it was not cross-reactive with other swine viruses.The critical value of negative and positive serum was 0.203 and the reproducibility was good.With the application of the indirect ELISA for PBoV established in this paper,the author collected and detected 90 swine serum samples from most regions in Jiangxi Province and found out the positive rate of detection was 68%(61/90).