| Infectious bovine rhinotracheitis(IBR)is an acute contact infectious disease caused by bovine infectious rhinotracheitis virus(IBRV)with severe respiratory and reproductive tract infection as the main symptoms.At present,the disease has been distributed all over the world and has a serious impact on the global cattle industry.Therefore,it is of great significance to establish a rapid and accurate serological diagnosis method for the prevention and treatment of IBR.In this study,gD protein was obtained through prokaryotic expression,and IBRV antibody detection test paper was successfully prepared by double antigen sandwich method.It also tried to prepare test paper by indirect method,so as to provide theoretical and technical support for the preparation of test paper for rapid clinical detection of ibrv antibody and other pathogenic antibodies.1.The IBRV gD gene was amplified by PCR and the expression vector p ET-32a-gD was constructed.After induced expression,the denatured gD protein was purified by nickel ion affinity chromatography.After renaturation and concentration,it was analyzed and identified.The results showed that the molecular weight of gD protein was about 53 k Da,and the mass concentration of purified and refolded protein was 4.02mg/ml.Western-blot and indirect ELISA showed that the protein had good reactivity,which laid a foundation for the later study of IBRV antibody detection test paper.2.The colloidal gold immunochromatographic test paper was successfully prepared with IBRV standard positive serum as the detection target,the prepared gold labeled gD protein coated on the binding pad as the detection probe,the gD protein coated on NC membrane as the detection line,and the gold labeled mouse Ig G and Sheep anti mouse Ig G as the independent quality control system.The test paper has good specificity and sensitivity,and the total coincidence rate with the imported commercial ELISA kit is96.7%.The test paper provides an effective tool for the qualitative detection of IBR in clinical serum.At the same time,an attempt was made to prepare colloidal gold immunochromatographic test paper by indirect method,but it was unsuccessful. |
