Studies On The Expression Of FMDV3AB Gene And The Rapid Detection Method Of3AB Antibody

Foot and mouth disease (FMD) is an acuate, febricity and highly contagious disease caused by Foot and mouth disease virus (FMDV). OIE as well as our country seted the disease as the first one of class-A infectious diseases.Our country mainly used vaccination to prevent the disease’s breaking. Vaccinated animals could also product antibodies of FMDV, in the condition of using vaccine, establishing a diagnostic method which could distinguish the vaccinated animals and the natural infected animals is an important topic of preventive technology of FMD. The vaccine used in our country is mainly destroy infectivity vaccine. During the process of producing vaccine, most non-struct proteins had been eliminated, so the vaccinated animals did not produce antibodies to the non-structural proteins. But the natural infected animals could express non-structural proteins, so it could produce the relative antibodies of FMDV. Thus we could distinguish the vaccinated animals and the natural infected animals by the way of detecting antibodies to the non-structural proteins. This article was mainly about establishing a diagnostic method which could differentiate the vaccinated animals froom the natural infected animals by the way of detecting antibodie to the non-structural protein3AB of FMDV.The3AB gene was inserted into vector pET-28a(+) and expressed efficiently in E.coli BL21with optimization condition. The protein was expressed mainly as inclusion bodies, the protein’s molecular weight was about33ku, The expressed products were purified through Ni+affinity chromatographic column. After purification, the purity of the protein was up to95%, and the purified protein was taken as antigen for the following trial.Using purified3AB protein as antigen to coat in the plate, established a diagnostic method of indirect ELISA by the detection of antibodies to the non-structural protein3AB. The optimal conditions were determined by square matrix method:the optimal concentration of antigen was0.625μg/ml, the optimal dilution of sera were1:100, The best working concentration of anti-pig IgG-HRP was1:25000. And the confining liquid, diluent, working time of serum and substrate was also been optimized. The judgement stander of the method was determined by referencing the judgement stander of imported kit. The results of specificness test, reproducibility test and stability test showed that the method established had obtained satisfactory result.The research also took use of Immune Colloidal gold technique (ICG), Using purified3AB protein as antigen, established the rapid diagnostic method by detecting antibody to the non-structural protein3AB of FMDV, including Dot immunogold filtration assay (DIGFA) and Colloid gold immunochromatographic assay (GICA). Synthesize colloidal gold particles by trisodium citrate disoxidation method, SPA was labelled with the colloidal gold, the colloidal gold labelled with SPA was purified by centrifuging, then diluted to the optimal concentration with diluent of colloidal gold. The research established DIGFA and GICA witch could differentiate the natural infected animals from vaccinated animals by the way of optimizing the concentration of antigen coated on the NC film, the confining liquid and the concentration of colloidal gold labelled with SPA. Both the methods had the merits such as operating convenient, did not need special environment and sample processing, the detection time was short (2-5min), the result showed directly and so on. So the method would take important application value on the rapid diagnosis of FMD.