Expression And Purification Of BCSP31 Protein From Brucella Melitensis And Preparation And Characterization Of Monoclonal Antibodies Against BCSP31

Brucellosis is one of the most common anthropozoonosis worldwide. As the causing agent of brucellosis and a facultative intracellular bacterium, Brucella consists of seven species according to antigenic variation and primary hosts, among which three species commonly infected both human and animals are Brucella melitensis, Brucella abortus and Brucella suis. Human and animals can be infected by Brucella through various routes, and manifested to be highly pathogenic, widely prevalent and severely harmful. In addtion, categorized in class II contagious agents according to Chinese Law of Contagious Diseases'Prevention and Treatment, Brucella is considered a potential bioterror agent as well. The clinical manifestation of Brucellosis varies greatly, making its diagnosis difficult and causing heavy economic losses if not treated properly. More importantly, the current detection assays for Brucella are far from satisfaction with regard to simplicity, rapidness and efficiency, especially on their low specificity, for example, hard to distinguish natural infection from vaccination. Thus, the development of faster, more sensitive and specific detecton methods against brucellosis is of much significance on prompt disposition of sick animals and proper treatment of sufferers.Researches have revealed that bacterial protein BCSP31 is a Brucella cell surface molecule dominating antigenicity and homogeneity. The present study aims to establish a repid, sensitive and specific ELISA assay to detect BCSP31 protein of Brucella melitensis by raising monoclonal antibodies against expressed BCSP31.Herein, the expression vector pGEX-4T-1-BCSP31 was transformed into E.coli, and purified proteins GST-BCSP31 and BCSP31 were obtained via affinity chromatography. The mAbs against BCSP31 were made by routine procedures: mouse immunization, cell fusion, cloning, ascites production and purification. The properties of these mAbs were analyzed by SDS-PAGE, Western-blot and ELISA.Results demonstrated that both GST-BCSP31 and BCSP31 were expressed in E. coli in soluble forms at 25℃, and further purified via affinity chromatography at about 31 kDa detected by Western blotting using rabbit antisera against Brucella melitensis. Two mAb hybridoma cell lines were obtained, designated 1F1 and 1E5 respectively, and both of IgG1 subclass. The results of epitope analysis via competitive ELISA and pairwise testing showed that the two mAbs recognized different epitopes, with higher relative affinity of 1F1 than 1E5. Specificity tests results of mAbs showed that they only reacted with BCSP31, instead of unrelated antigens such as HCV AS3, Mycobacterium tuberculosis DAF 44a and RPF, Hantan virus GP and NP and human IgG1 as well as E.coli lysates.The optimized working conditions of indirect sandwich ELISA is as such: coating of 10μg/mL mAbs in 100μl per well, 20μg/mL of antigen in 100μl per well, and 1:50000 diluted HRP-labelled goat anti-rabbit IgG in 100μl per well. The ELISA assay exhibited fairly good specificity, sensitivity and reproducibility.To conclude, the establishment of an ELISA assay based upon two mAbs against expressed BCSP31 protein would help us understand Brucella antigens pertaining to infection, immunization, and pathogenesis, and meanwhile pave the way for the development of an ELISA detection kit for Brucella.