Fuctional Study On BsIA Adhesion Of Bacillus Anthracis

Bacillus anthracis, is the causative agent of anthrax in humans and animals. Human infection occurs when endospores enter the body through abrasions in the skin or by ingestion or inhalation. Many researches show that separate PA protein vaccine can’t provide enough immunoprotection. Presently, many researchers consider that PA plus other bacilli and spore components is a promising tactics for anthrax vaccine researching, therefore, researchers have been searching and identifying new protective antigens of B. anthracisThe S-layer of B. anthracis is made up of protein arrays that self assemble into a two-dimensional crystalline sheet around the outside of the cell envelope and its function is as protective outer membrane, the molecular, adhesion and so on. Bs1A is a surface layer (S-layer) protein and a SLH domain containing protein and its gene bs1A (pXO1-90) is located within the pXO1plasmid that also encodes for toxin genes. A more recent study showed that indeed, Bs1A is required to mediate adherence between host cells and vegetative forms of encapsulated bacteria. Therefore this project intends to in-depth study of Bs1A.The first part works uses PCR to amplified the Bs1A gene fragments, constructing Prokaryotic expression vector, recombinant protein separation and purification, preparing Polyclonal Antibody and identifying the function of Bs1A protein through fluorescence-activated cell sorting, bacterial adhesion inhibition experiments, and immunofluorescence experiments. From the results of the experiment, bacterial strain pET28a-Bs1A/Rosetta(DE3) and corresponded soluble expression were constructed and reconstructed successfully. The purity of more than87%of the Bs1A protein is got through ProBondTM purification system. Flow cytometry and immunofluorescence results show that protein Bs1A has adhesion function. Bacterial adhesion inhibition experiments showed that Bs1A protein may be a potential protective antigen.The second part of the experiment uses protein structure prediction software to determine the location of the core functional domains may exist, then the fragmental research was processed. In order to do better and more convenient detection, we fuse the protein fragments were fused with the EGFP proteinto identify the fragmental function of protein Bs1A. The results show that pET28a-EGFP/Rosetta(DE3)and corresponded soluble expression and purification. From the effect of expression and flow cytometry, constructed EGFP protein has great fluorescence. Also, according to the prediction of protein structure software we divided the first fragments of protein Bs1A and pET28a-EGFP-Bs1A (260-500)/Rosetta (DE3), pET28a-EGFP-BslA(411-560)/Rosetta (DE3), pET28a-EGFP-Bs1A(511-652)/Rosetta (DE3) and corresponded expression and purification were constructed successfully. Fluorescence electron microscopy results showed that the fragment BslA (260-500), BslA (411-560) have adhesion function, so the core domain function is located of411-500amino acids of BslA protein. According to the results, the second dividing was processed and pET28a-EGFP-BslA (411-455)/Rosetta (DE3)、pET28a-EGFP-BslA(431-485)/Rosetta (DE3). pET28a-EGFP-BslA (456-500)/Rosetta (DE3) and corresponded expression and purification were constructed successfully. Immunofluorescence results showed that the fragments BslA(411-455), BslA (431-485) have adhesion function, which define the core functional domain is located at431-455sites of BslA protein.In conclusion, BslA is an important protective antigen, and may play an important role in the research and development of next-generation anthrax vaccine. The determination of core functional domain of protein BslA laid a good foundation to further researching B. anthracis pathogenic mechanisms and immunization.