Tandemexpression Protein A/G/L Ig Immunoglobulin Binding Domain And Application For Foot-and-mouth Disease Distinguish Diagnosis

Staphylococcus aureus protein A(Staphylococcal protein A, SPA), Streptococcus(C and G group)protein G(Streptococcal protein G, SPG) and Peptostreptococcus protein, L(Peptostreptococcus protein L, PPL) are all typical immunoglobulin-binding proteins, which can bind immunoglobulins of most mammals, but their binding way and combination of capacity with IgG from different species are different. They make their respective advantages complementary to each other, which make them not only have a better combination of capacity, but also have a broader spectrum combination, these characteristics could make them have a good prospects in application.In this study, PPL B3 domain was optimized by bioinformatics software, then the gene tandem was conducted to form different copy of AGL with the existing AG in our laboratory. The tandem genes were cloned into prokaryotic expression vector pET-30a(+) to conduct the recombinant expression vector pET-30a(+)-L, pET-30a(+)-AGL, pET-30a(+)-[AGL]2 and p ET-30a(+)-[AGL]3.After transformed into Rosetta(DE3)PlysS, recombinant proteins L, AGL, [AGL]2 and [AGL]3 were expressed, which were all expressed as the soluble form, and AGL, [AGL]2 and [AGL]3 protein have specific reaction with bovine serum. After purified by Ni-NTA affinity chromatography, we found recombinant protein [AGL]2 has high expression and purification. Western blot and indirect ELISA analysis showed recombinant protein [AGL]2 can react with nine kinds of mammals Ig, which has stronger binding capacity than recombinant proteins [AG]3. After labelled by HRP, we found it has a high binding activity with rabbit IgG. Dot-ELISA and indirect ELISA analysis showed that[AGL]2-HRP could bind mammals immunoglobulins from nine species, and it also has stronger binding activity than [AG]4-HRP in binding human IgM and IgA. [AGL]2-HRP could also be used to detect rabbit immune antibody; As the secondary antibody in detecting brucellosis antibody in bovine,dog, deer, swine sheep serum and bovine viral diarrhea virus(BVDV) antibody. On the basis of8BF-ELISA for detecting FMDV antibody established in our laboratory, we determined the optimal reaction conditions and cut-off vaule for AGL-ELISA to detect the cattle, pig, sheep and deer serum.We used the optimal AGL-ELISA to detecting the serum from 12 farms including 904 cattle, 174 pig,292 sheep and 80 deer, the positive rate ranged from 0 to 76.19%.