牛传染性鼻气管炎抗体胶体金免疫层析方法的建立-兽医学专都业毕业论文.docx

Establishment Establishment of Gold Immunochromatographie method for Infectious Bovine Rhinotracheitis Antibodies Author：LiuTeng Professor：Prof．Zhao Yue．Lan Major：VeterinaryMedicine Abstract Infectious bovine rhinotracheitis(IBR)is caused by infectious bovine rrhinotracheitis virus(IBRV) (Bovine herpesvirus一1，BHV-I)and known mainly acute，pyrexic and contagious disease of respiratory tract which is characterized by rhinotracheitis，conjunctivitis，fever’abortion and infectious pustular vulvovaginitis．For the field detection of the disease，the existing detection method has some deficiency,so to establish rapid，simple and accurate detection method is very necessary． In this study,the major antigens of bovine rhinotracheitis virus，ga Was analysised by DNAstar． The epitope gBa，gab and gBc which is in hi【gh antigenic index was Synthesised and obtained the fusion gene PUCK—gaabc by linker．Then the fusion gene was inserted into the pET028a(+)to construct the recombinant plasmid pET28a-gBabc．The recombinant plasmid was transformed into Escherichia coli BL21(DE3)to express the fusion protein which was induced with IPTG．The expressed fusion protein was in soluble in SDSanalysis and Western-blot detection．These resules demonstrated that the purified protein was 44．3 kDa(include 3．5 kDa pET-28a(+)in molecular weight)． The purified bovine recombinant protein gaF fcontaining final concentration of O，6mg／mL)of infectious bovine rrhinotracheitis virus was immobilized nitrocellulose membrane detected line antigen，rabbit IgG(containing final concentration of 1．5 mg／mL)was immobilized nitrocellulose membrane control line as antibody,colloidal gold labeled SPA was sprayed glass cellulose membrane to prepare the colloidal gold immunochromatograpic strip．There was red reaction strip at the test line only when detecting the standard positive selum，and the red reaction strips all appeared at the control line．The SPA was labeled with the colloidal gold solution of 1 5NM，and the marker PH was