Establishment Of Indirect ELISA Method And Crystal Culture Based On The H Protein Of Peste Des Petits Ruminants Virus

Peste des petits ruminants(PPR)is an acute and contagious viral disease affected small ruminants(i.e.,sheep and goats),with the morbidity and mortality of more than 90%.The causative agent of this disease,PPR virus(PPRV),is classified into the genus measles virus of the family Paramyxoviridae.PPR is a must reported disease listed by the World Organization for Animal Health(OIE),and it is also a type of animal disease regulated by my country.At present,there is no effective treatment for PPR,which mainly relies on vaccination for prevention and control.Therefore,it is necessary to establish accurate and rapid antibody detection methods to evaluate the immune effect of vaccines.H protein is a structural protein that can stimulate the body to produce neutralizing antibodies,and has the potential to establish an ELISA antibody detection method.Protein function mainly depends on spatial structure,so obtaining accurate protein structure information is very important for studying protein function.At present,the protein structure of some viruses in the Paramyxoviridae family has been resolved,but the H protein structure of Peste des petits ruminants had few reported.In this study,the H protein gene of PPR Nigeria/75/1 strain was synthesized,and the recombinant target protein was obtained by yeast expression and purified.The crystal culture conditions of PPRV H protein were explored.In addition,the purified H protein was used as the coating antigen to establish an indirect ELISA method for the detection of PPRV antibodies.Then this study verified the specificity,sensitivity and reproducibility of the method and compared with ID Screen? PPR Competition.Obtained the following research results::1.The pGAPZ?-PPRV-H recombinant plasmid was successfully constructed and the recombinant protein was obtained.The H protein gene of Nigeria/75/1 strain was codon-optimized and His tag was added,and it was successfully constructed into the pGAPZ? vector.Transform into SMD1168 engineered bacteria for Pichia pastoris expression to obtain recombinant protein.Purify the target protein with imidazole-containing solutions of different concentrations.After SDS verification,the expressed target protein is correct in size.Western Blot results show that the expressed target protein can specifically react with PPRV positive serum and has good reactogenicity.2.Preliminary acquisition of PPRV-H protein crystal culture conditions.PPRV-H protein was available at 1 M BIS-TRIS p H 7.5,25% w/v polyethylene glycol 3350;0.1 M potassium thiocyanate,30%w/v polyethylene glycol monomethyl ether 2000;0.1 M HEPES p H 7.5,0.2 M lithium sulfate The monohydrate,25% w/v polyethylene glycol 3350 initially formed crystals.3.Successfully established PPRV indirect ELISA antibody detection method.The antigen coating concentration was 5 ?g/m L;the blocking solution was 1% casein,and the condition was 37°C for 2 h;the dilution of the serum to be tested was 1:5,and the reaction condition was 37°C incubation for 30minutes;the dilution of the enzyme-labeled secondary antibody The reaction condition was 1:3000,and the reaction condition was 37°C for 30 minutes;the color solution reaction condition was 37°C for 10minutes;the critical value was determined to be 0.320.After determining the basic conditions,the method was tested for specificity,sensitivity and repeatability.The results showed that the method had no specific reaction with the positive sera of sheeppox virus,sheep bluetongue virus,Brucella,ovine oral disease virus,and foot-and-mouth disease virus;the standard positive serum can still detect positive when diluted to 1:800.Sensitivity is good;the coefficient of variation between batches and within batches was within 8.9%;the compliance rate with ID-vet standard detection kit was 93.97%.