Expression Of Ag85B Gene Of Mycobacterium Bovis And Development Of Indirect ELISA

Bovine tuberculosis (BT) is a chronic wasting disease primary caused by Mycobacterium bovis that can infect human and a lot of animal, bovine is the animal of the most sensitive, especially the cow. According to the investigation, more than 10% human tuberculosis are aroused by bovine tuberculosis, therefore Mycobacterium bovis is the important disease source of human tuberculosis, so we get hold of the quarantine method of high sensitive and high accuracy rate is to prevent people infect the Mycobacterium bovis. In this paper, Ag85B gene of Mycobacterium Bovis is expressed and indirect ELISA is developed to detect bovine tuberculosis.We according to the Ag85B gene sequence published in GeneBank of AF2122/97 strain, primers were designed, the DNA fragment of Ag85B gene was amplified by PCR from Mycobacterium bovis Valleeâ…¢,the purity product was cloned initially into PMD18-T,then positive colony was choosed and sequenced and the fragments were inserted into the orientation of the vector pET-32a (+),by Western-Blot detection , the protein was have been expressed successfully, Expressed protein of high purity were purified by His-bind chromatography at last.In this experimentation we also set up Ag85B-ELISA procedure with purified Ag85B protein as coating antigen,then we choose the best condition for our experimentation such as diluents for serum ,blocking buffer ,incubation time for enzyme-substrate reaction and the method of result interpretation and so on .At last we do comparison Ag85B-ELISA and PPD-ELISA at three aspects coincidence,sensibility,particularity,the result show that PPD-ELISA has more sensibility than Ag85B-ELISA,and Ag85B-ELISA has more particularity than PPD-ELISA ,because the experiential animals were so little that the coincidence effect was not distinct.