Detection Of Bovine Rotavirus By RT-PCR And Establishment Of Indirect ELISA To Detect Bovine Rotavirus Antibody With VP6 Protein

Brovine Rotavirus (BRV) characterized as a member of the reoviridae. Rotavirus is a main pathogeny that can cause brovine serious diarrheas and cause worldwide economic losses in graziery.In this study, we designed a pair of primers which between the guarded segment (513~919), and got about 400bp segment by extracting the nucleic acid from cell culture and fecal samples. The result of sequencing indicated the gene was got from VP6, and the result of sensation indicated that the result of PCR is positive, when the virus content in cell culture is 10-3TCID50. And the primers also can get the VP6 gene from PRV, but can not get the result form TGEV, PEDV. The results show that the methods has a highly specificity to A group reoviridae. Detection of annealing temperature indicates that the temperature between 40℃and 60℃have no affection to the result. The results of the study indicated the diagnostic primers have a certain value.Recording to the reported VP6 nucleotide sequences from NCDV GenBank, a pair of primers was designed to amplify VP6 gene of NCDV by oligo6.0. There recombine plasmid pProHTa-VP6 was transformed into Rosetta and induced, then identified, analyzed the expressed products by SDS-PAGE and western-blot, the results showed that a protein about 52kDa was expressed. Purified the recombined protein by Ni affinity chromatography, and got the recombinant VP6 protein. The analysis by western-blot showed that the proteins had a positive reaction with anti-NCDV serum. The VP6 protein is a cheaper antigen than virus, which can avoid the troubles that came from culturing virus, purified virus.Initial establishment indirect ELISA detected bovine rotavirus antibody with VP6 protein. The experiment searched the conditions about concentration of santigen, the condition coating, sealing.The experiment used of orthogonal experiment with square indirect ELISA method to determine the best conditions: coating antigen is diluted with pH8.6 phosphate to 0.025μg/mL hole, and coated at 37℃still 2.0h or at 4℃for a night for 250μl/hole, washed with PBST 3min at three times, and then took into 4%PEG20000 for blocking fluid as 200μl/hole, blocked at 37℃at 2h, washed with PBST 3min at three times, and then took into serums diluted with 1:100 as 100μl/hole at 37℃still 1.0h, washed with PBST 3min at three times, and then took into HRP as 100μl/hole, reacted at 37℃still 1.0h. Colorated in dark place for 10min to 15min, took into 2mol/mL H2SO4 50μl/hole to end the reaction. Measurtement OD492nm with instruement of enzyme.