Cloning,Expression Of Bacillus Anthracis BclA Gene And Preparation Of Monoclonal Antibodies Against BclA Protein

Bacillus anthracis , the aetiological agent of anthrax , was the first discovered pathogenic bacterium in history Rapid progresses have been made on this field in recent years , especially ; Bacillus anthracis has been sequenced successesfully in 2004 and published on the Internet.Bacillus anhracis collagen-like protein(BclA) is a structural component of the exosporium filaments,as well as the imumunodomainant antigen on the spore surface. The genes encoding BclA proteins of were cloned and sequenced from an Bacillus anthracis stain separated from China. polymorphism of the Bacillus anthracis BclA protein may tell us the difference between different stains of bacillus anthracis. To facilitate investigation into BclA protein, BclA gene of 1119bp was amplified by high fidelity PCR from strain which was separated by the member of our lab. The PCR product was digested by restriction enzymes and then subcloned into the prokaryotic expression vector pGEX-6p-1. The GST- BclA fusion protein of about 68KUwas expressed in BL21 E.coli after IPTG induction, which was revealed by SDS-PAGE .The fusion protein was purified by Glutathione Sephrose 4B column and used to immunize BALB/c mice. SP2/0 myeloma cells and spleen cells of the immunized mice were fused by PEG-1000. The hybridoma culture supernatants were screened for BclA -specific antibody production .The result of Western blotting showed that the three monoclonal antibodies reacted positively with the GST fusion protein and not with the GST carrier protein expressed in E.coli.