Prokaryotic Expression Of Infectious Bovine Rhinotracheitis Virus GB And GE Gene Protein And Establishment Of Indirect Elisa For Detection

Infectious rhinotracheitis in cattle affects the normal growth of the herd,makes the breeding cycle longer and reduces resistance,which can stimulate other diseases.It causes huge economic losses and to a certain extent hinders the flourishing of the cattle industry,as well as having a bad impact on the international trade of our animals and animal products.The recommended serological methods for the detection of bovine infectious rhinotracheitis in China are the virus neutralisation test and the ELISA method.The virus neutralisation test is time-consuming,labour-intensive and subject to objective factors,and is not suitable for the detection of bovine infectious rhinotracheitis virus in China,let alone for epidemiological investigations.However,they are expensive and not suitable for clinical application in China.Therefore the development of a successful ELISA kit is extremely important.In this thesis,the main antigenic regions of bovine infectious rhinotracheitis gB and gE were cloned,expressed in prokaryotic and eukaryotic form,and an indirect ELISA assay for detecting antibodies to the virus was established for the expression of the purified recombinant proteins,providing an auxiliary technical tool for the development of an IBR diagnostic method with good application prospects for social needs,and the establishment of the gE-ELISA method can be used to distinguish The method can be used to differentiate natural infection from wild strain infection.The results of the study are as follows:Expression fragments of the gB(312-529 aa,661 bp)and gE genes(188-420 aa,667 bp)of bovine infectious rhinotracheal virus were successfully cloned and analysed by DNASTAR Biosoft.The amino acids deduced from these two genes had major epitope antigens and did not contain signal peptides with no transmembrane regions.The recombinant prokaryotic expressionplasmids p ET-32a-gB and p ET-28a-gE were constructed and transferred into BL21.The two recombinant proteins were expressed mainly as inclusion bodies by SDS?PAGE and purified by affinity chromatography on a nickel column to obtain a large amount of high purity 60 ku gB protein and 30 ku gE protein,respectively.Western blot analysis showed that the two recombinant proteins were able to bind specifically to IBRV positive sera and had some reactogenicity.An indirect ELISA assay for bovine infectious rhinotracheitis was established using purified gB protein as the encapsulated antigen.The optimal encapsulation volume was 8 ?g,and the sera to be tested were diluted at 1:200 and the secondary antibody was diluted at 1:2 500.The method did not cross-react with sera positive for bovine herpesvirus type 3,bovine coronavirus and bovine rotavirus.Comparison with the commercial IBR gE-ELISA kit from IDvet showed a positive compliance rate of 86.5%.An indirect ELISA assay for bovine infectious rhinotracheitis was established using purified gE protein as the encapsulated antigen.The optimal encapsulation volume was 5 ?g,the sera to be tested were diluted at 1:25 and the secondary antibody was diluted at 1:2 500.The method did not cross-react with sera positive for bovine herpesvirus type 3,bovine coronavirus and bovine rotavirus.Comparison with the commercial IBR gE-ELISA kit from IDvet showed a positive compliance rate of 92%.