Prokaryotic Expression of Recombinant VP6*Protein of Porcine Rotavirus and Establishment of Indirect ELISA Detection Method
This study was designed to establish an antibody detection method for porcine rotavirus(PoRV).We utilized the truncated VP6*(aa149-332)protein of PoRV as the detection antigen to develop an indirect ELISA detection method for PoRV antibodies.The truncated recombinant VP6*(aa149-332)exists in two forms:inclusion bodies and soluble,with a size of 22.5 ku.The optimized reaction conditions of the ELISA were as follows:the optimal coating antigen concentration,coating time and temperature were found to be 25 ng per well,and left overnight at 4 ℃;the plates were blocked with 5％skimmed milk at 37 ℃ for 2 hours;the optimal dilution of test serum and the secondary antibody were found to be 1∶400 and 1∶24 000,respectively.The optimal incubation time and temperature for test serum and the secondary antibody were 30 minutes and 37 ℃,respectively,and the color development time was 15 minutes.Testing 30 negative sera determined the cutoff value at OD450 nm=0.418,the maximum dilution of detectable antibodies is 1∶3 200.The intra-batch and inter-batch coefficient of variation were both less than 10％,and it exhibited good specificity.The agreement rate with Western Blot results for 28 sera was 96.42％.The correlation coefficient(R2)between ELISA OD450 nm values and neutralizing antibody log2 values was 0.878 5.In summary,the indirect ELISA established in this study for detecting porcine rotavirus antibodies has high specificity and sensitivity,making it applicable for clinical sample testing of porcine rotavirus.
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维普
