Preparation For The Detection Reagents And Development Of The Methods For Rapid Diagnosis In Transmissible Gastroenteritis

E.coli DH5a harbouring recombinant plasmid pProExHTb-N was induced with IPTG to express the recombinant nucleoprotein (rNP) of transmissible gastroenteritis virus (TGEV).The rNP collected after cell extraction was applied onto the nickel nitrilo-tri-acetic acid (Ni-NTA) resin. The optimized procedure of purification was lysing the E.coli by Buffer A (containing Immol/LPMSF) .washing the resin with 2+Buffer A (pH8.0),2+BufferB (pH6.3), 2X BufferC (pH5.9) and collecting the elution of Buffer C (80mmol/L imidazole) .The molecular weight of purified rNP was 47ku analyzed by SDS-PAGE.The result of Western-Blot showed that the rNP can act to sera of anti-TGEV. The concentration of the elution including rNP was identified 154.Vug per ml-4V9.Vug per ml by Bradford's assay, at the same ratio 1.52mg rNP can be abtained per gram E.coli sedimentDot-ELISA and indirect-ELISA were established by the rNP. The optimal amount of the rNP coated in Dot-ELISA was 1.25g per dot. The pre-dot membrane can be kept 5 weeks long at 4 C. The specific assay showed that rNP have no cross-reaction with porcine epidemic diarrhea coronavirus (PEDV) porcine rotavirus (PRV) .In indirect-ELISA the optimal amount of coated antigen was 0.5lg per well,the optimal blocking solution was 5% dry milk,the ideal dilution of serum was 200 and the fit reaction time was 30mins the adequatable reacrion time of substrate was 10min.The monoclonal antibody (McAb) detecting TGEV was performed by immunizing the BALB/c mice with purified rNP. Cells fusion was carried out by standard method and hybridmas with rNP-antibody-positive supernatants were subcloned third by the limiting dilution. One hybridoma cell line secreting monoclonal antibody specific against rPN was picked out by indirect-ELISA designated E10F10D5. The even number of chromosomes per hybridoma was 8V +10 paires .The antibody liter of ascites v/as 1:12800 determined by indirect-ELISA. TGEV was applicated as antigen in Western-Blot and indirect-ELISA to analyze the McAb. The result showed that the McAb could indetified certain antigenic epitope of TGEV N protein. Method to dispose TGEV in intestinal sample was optimized.Two methods to detect antibodies of TGEV were established firstly by using rNp as antigen coated in Dot-ELISA and indirect-ELISA in domestic. One hybridoma cell line secreting monoclonal antibody recognizing TGEV N protein was produced. All the work founded material base for TGE diagnosis accurately and rapidly.Postgraduate: QianJiang Major: Preventive Veterinary ScienceSupervisor: Professor Li YiJing...