Establishment And Preliminary Application Of Indirect ELISA Based On BVDV E2 Gene

Bovine viral diarrhoea virus(BVDV)is an important pathogen that affects the economic development of the cattle industry,causing bovine viral diarrhoea(BVD).BVD is an acute and reproductive disorder infectious disease that causes symptoms such as viral diarrhea,fever,mucosal,ulceration,and decreased white blood cells.The developed countries through the detection and isolation of PI to control BVD,the incidence of genotype in Henan,Shandong and Xinjiang and other regions of different BVD type BVDV-1 was the predominant genotype,early detection and early diagnosis and early treatment is the key to control the disease.In this study,BVDV was used as the research object,the E2 gene was cloned,expressed,purified and identified,and the indirect ELISA detection method of BVDV was established.1 Prokaryotic expression and purification of BVDV E2 gene encoding proteinE2 gene was amplified from BVDV by PCR method,And the recombinant plasmid p ET28a-E2 was constructed by ligating the prokaryotic expression vector p ET28 a,1mmol/L IPTG induced expression of E2 protein,SDS-PAGE electrophoresis,and affinity purified by Ni-NTA column chromatography,the soluble expression and purification of the natural conditions,used the different concentration of imidazole washing impurity protein,inclusion bodies were purified under denaturing conditions,used different salt concentrations of 8M urea to wash the impurity protein.The purified protein by Western blot identification and analysis of recativity.Result: The successful construction of p ET28a-E2,and expressed in Escherichia coli,expression the molecular weight of the product is about 42 KDa,the purified protein concentration is 16.7 ?g/m L and can be BVDV positive sera,have good recativity.Conclution:The expression of E2 gene was successfully established,which laid the foundation for the establishment of BVDV detection method.2 The establishment and preliminary application of indirect ELISA detection method of BVDV E2 geneThis test determines the optimal concentration of antigen by determining the optimal dilution of serum,the optimal blocking solution and blocking time.the optimal Rabbit anti-bovine Ig G/HRP concentration and reaction time and substrate reaction time.After determining the critical value,the specificity and sensitivity and repeatability test were used to illustrate the effect of establishing the ELISA method.Result: concentration of antigen 1:32 and dilution of serum1:100,5% skim milk closed for 90 min,Rabbit anti-bovine Ig G/HRP antibody1:3200 dilution for 60 min,substrate for 10 min,P/N value is the largest,negative critical valueof 0.259,with the commercialization of the total compliance rate of 90.9%,The indirect detection method of BVDV E2 gene was established.After application,it was confirmed that the established indirect ELISA method had better specificity and sensitivity.