Screening and evaluation of antigens for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in naturally infected young sheep
Objective To analyze the antibody responses of 10 serum reactive antigens(MAP1 138c,MAP2121c,MAP0150c,MAP0862,MAP0209c,MAP2120c,MAP0038,MAP3420c,MAP 2154c and MAP2751)of Mycobacterium avium subspe-cies paratuberculosis(MAP)in naturally infected young sheep and evaluate their diagnostic value.Methods Serum sam-ples and anal swab samples were collected from 6-month-old sheep without obvious PTB symptoms in the flocks with paratu-berculosis(PTB)history.The serum samples were tested by PTB antibody detection ELISA kit,and anal swab samples were detected by the fluorescent quantitative PCR based on MAP F57 element.The sheep were grouped according to the test results.PCR was used to detect 10 MAP antigen genes in positive anal swabs.The antigen genes were cloned into pET-28a and induced to be expressed in E.coli BL21(DE3)strain by IPTG.The recombinant antigens were purified by Ni-Sepha-rose,and then coated on the ELISA plate for testing the collected serum samples to analyze the antibody reaction of the selected antigens in naturally infected sheep.The detection rates of serum antibodies against different antigens were analyzed to evaluate the diagnostic value of the antigens.Results The 72 sheep sampled were divided into three groups:anal swab positive-antibody positive(n=34),anal swab positive-antibody negative(n=23),and anal swab negative-antibody nega-tive(n=15).All 10 antigen genes were detected from positive anal swabs,and sequences of each gene were highly consis-tent.Through ELISA detection,MAP1138c,MAP2121c,MAP0150c and MAP0862 produced antibody reactions in infe-cted sheep.Antibodies against MAP1138c,MAP2121c,MAP0150c and MAP0862 were detected in 30,34,24 and 31 of the 57 infected sheep,respectively.In the anal swab positive-antibody positive group,the detection rate of anti-MAP1138c antibody was the highest(76.47％).In the anal swab positive-antibody negative group,the detection rates of antibodies against MAP2121 and MAP0862(52.17％and 47.83％)were higher than those of the other two proteins.In the detection of 72 serum samples,the overlap of ELISA coated with MAP2121c and MAP0862 exceeded 91％.Conclusion MAP1138c,MAP-2121c and MAP0862 may be dominant biomarkers to induce MAP antibody response in naturally infected young sheep.MAP2121c and MAP0862 can make up for the deficiency of sensitivity of commercial ELISA kits in early diagnosis of PTB.
NETL
NSTL
万方数据
