| Since April 2010,a viral infectious disease of ducks,mainly characterized by decreased egg production and even extinction,has been occurring in breeding ducks and laying ducks in most duck breeding areas in China,accompanied by weight loss,decreased appetite,high fever and other symptoms.The disease spread widely and caused huge economic losses to the duck industry in China.The NS1 protein,the first non-structural protein of TMUV,is antigenic and can be used for the preparation of vaccines and the monitoring of antibody levels after immunization;at the same time,the NS1 protein is the only secreted protein of the TMUV virus protein and can be used for the diagnosis of TMUV infection.Primers were designed based on the sequence of NS1 gene of TMUV CQW1 strain,and the prokaryotic expression plasmid p ET-32a-NS1 was constructed by homologous recombination,and the NS1 prokaryotic expression protein was obtained by induction of expression with IPTG using E.coli BL21 expression bacteria.After a series of optimization to determine the IPTG induction concentration,time and temperature,the optimal induction conditions were determined to be for 6 h at 37°C with the addition of 0.4 mmol/L IPTG concentration.Subsequently,the heavily expressed recombinant NS1 protein inclusion bodies were purified by SDS-PAGE gel cutting.The purified recombinant NS1 protein was immunized in mice and ducklings,and mouse anti-TMUV NS1 polyclonal serum and duck anti-TMUV NS1 polyclonal serum were prepared.Finally,after Western Blotting and IFA identification,it was determined that the prepared anti-TMUV NS1 polyclonal serum could specifically bind to TMUV NS1 protein.An indirect ELISA method was developed for the detection of TMUV NS1 antibody using purified TMUV NS1 protein as the encapsulated protein,and the optimal detection conditions were optimized and determined as follows: the encapsulated concentration of NS1 protein was 3.13 μg/m L;coated at 37℃ for 1 h;blocked at 37℃ for 1 h;duck antiTMUV NS1 polyclonal serum dilution was 1:32000,and the reaction time was at 37℃ for0.5 h;the dilution of HRP-labeled Goat anti-duck antibody was 1:500,and the reaction time was at 37°C for 1.5 h;the color development condition was at 37°C for 15 min.Then,the established indirect ELISA method for detecting TMUV NS1 antibodies was applied to detect positive ducks infected with duck hepatitis virus type I,duck plague virus,Salmonella,Escherichia coli and Riemerella anatipestifer.The results were all negative.This assay can detect NS1 antibodies in TMUV duck positive sera up to 1:3200 dilution and intra-and interbatch repeat coefficients of variation were less than 10%.After that,this thesis compared the established indirect ELISA assay for the detection of TMUV NS1 antibodies with the neutralizing antibody assay,and the compliance rate was 100%.Further,the established indirect ELISA for detection of TMUV NS1 antibodies was applied to detect artificially infected duckling sera with TMUV and positivity rate was 95.8%.A double antibody sandwich ELISA method for the detection of TMUV NS1 antigen was developed using mouse anti-TMUV NS1 polyclonal serum as the capture antibody and duck anti-TMUV NS1 polyclonal serum as the detection antibody,the assay conditions were optimized and the optimal assay conditions were determined as follows: mouse anti-TMUV NS1 polyclonal serum was diluted at 1:2000,coated overnight at 4℃;blocked at 37℃ for1.5 h;antigen reaction conditions were at 37℃ for 2 h;duck anti-TMUV NS1 polyclonal serum was diluted at 1:8000 and reacted at 37℃ for 2 h;HRP-labeled Goat anti-duck antibody was diluted at 1:750 and reacted at 37℃ for 2 h;color development time was for20 min at 37℃.The ELISA method was used to detect duck positive sera for duck hepatitis virus type I,duck plague virus,Salmonella,Escherichia coli and Riemerella anatipestifer,the results were all negative.This assay can detect NS1 antigen in TMUV duck positive sera up to 1:3200 dilution and intra-and inter-batch repeat coefficients of variation were less than10%.Finally,the established double antibody sandwich ELISA for detection of TMUV NS1 antigen was applied to detect artificially infected duckling sera with TMUV and the positivity rate was 99.0%.In conclusion,the indirect ELISA method for the detection of TMUV NS1 antibody and the double antibody sandwich ELISA method for the detection of TMUV NS1 antigen established in this thesis have good specificity,sensitivity and reproducibility.The results of this thesis provide a good laboratory serological assay for the rapid detection of TMUV infection. |
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