Study On The Important Immunogenic Proteins Of The Bacillus Anthracis

Bacillus anthracis is an acute zoonotic infectious disease. It is the etiological agent of anthrax leading to deaths. The virulence, regulatory networks and immunogenicity of the vaccine candidate were currently a hot research. Study of Bacillus anthracis vaccine and new candidate vaccine proteins have theory significance and applied value.Researchers needed to find new protective protein vaccine candidates.In this study, we cloned, expressed, purified and producted polyclonal antibodies of 15 proteins including BA0080, BA0309, BA0344, BA0345, BA1511, BA2551, BA4499, BA5565, BA5580, BA5588, BA1021, BA5017, BA5147, BA3338 and BA0887 of B. anthracis vaccine strain A16R. Western blot was used to validate the immunogenicity. The following are main results.15 genes have been amplified by PCR using the design primers and getting 15 purified fragments,these are 2430 bp,1542 bp,1521 bp,588 bp,1281 bp,783 bp,606 bp,606 bp,852 bp,846 bp,795 bp,1194 bp,1371 bp,453 bp,1995 bp. Constructed of the 15 genes in the prokaryotic expression vector;getting 15 recombinant bacterial pET32a-BA0080/BL21,pET32a-BA0309/BL21,pET32a-BA0344/BL21,pET32a-BA0345/BL21,pET32a-BA1511/BL21,pET32a-BA2551/BL21,pET32a-BA4499/BL21,pET32a-BA5565/BL21,pET32a-BA5580/BL21,pET32a-BA5588/BL21,pET32a-BA1021/BL21,pET32a-BA5017/BL21,pET32a-BA5147/BL21,pET32a-BA3338/BL21,pET32a-BA0887/BL21. Expressed of the 15recombinant proteins with the condition1 mmol/L IPTG,37℃,220 r/min, these proteins molecular weight are 90.2 kD,56.1 kD,54.5 kD,20.5 kD,47.3 kD,28.2 kD,22.4 kD,22.2 kD,30.4 kD,31.1 kD,31.2 kD,43 kD,52.8 kD,17.3 kD,70.4 kD. Purified of 15 recombinant proteins using ?KTA system uing the condition 1 mmol/L IPTG,20℃,180 r/min to induced 15 recombinant bacterial. Getting 15 polyclonal antibodies by immuned mice and these antibody titer are 1:12800,1:3200,1:3200,1:16200,1:48600,1:16200,1:16000,1:16200,1:16200,1:16200,1:16200,1:5400,1:16200,1:8000,1:16000. Western blotting identified the immunity of 15 proteins. Flow cytometry and Immunofluorescence defined the location of BA3338 protein on the cell wall.The result showed that these 15 proteins were proved to be immunogenic proteins and will provide new targets for new vaccine development against anthrax. Putative S-layer protein BA3338 was locatied in the cell wall.