Construction Of BA2380 Gene Deletion Mutant And Reventant Mutant Of Bacillus Anthracis

Bacillus anthracis,a virulent pathogen bacterium,is the causative agent of zoonotic acute infectious diseases,posed a serious threat to human and animal husbandry all over the word.The main form of anthrax spread and infection is spore,it was caused skin anthrax,intestinal anthrax,pulmonary anthrax and other diseases after infected anthrax.B.anthracis was used as a biological weapon to reserve.Therefore,the pathogenesis of B.anthracis has been paid high attention.The preliminary experiments in our lab showed that BA2380 alkaline serine protease was associated with the virulence factor and S-layer protein degradation of B.anthracis,was involved in the pathogenicity of B.anthracis infection,so it was necessary to conduct the in depth reaserch to BA2380 gene function.In this work,we constructed the BA2380 gene deleted mutant and reventant complementation mutant using B.anthracis A16D2 as the starting strain,and studied the physiological functions of the two mutants.First,the upstream and downstream homologous arms of the BA2380 gene and the spc fragment of the resistance gene were obtained by PCR amplification,and linking to the T-easy vector respectively,three donor plasmids were constructed.Three fragments were simultaneously inserted into the thermo-sensitive shuttle vector p KMBK using the "Golden Gate" cloning system,the gene targeting plasmid pK2380 usd was constructed.Then the gene targeting vector was electroporated into A16D2 competent cells of B.anthracis.Through homologous recombination,A16D2?BA2380::spc mutant with deleted BA2380 gene were screened,and subjected to a validation.Secondly,the BA2380 gene was amplified by PCR from B.anthracis A16D2 strain and introduced into Escherichia coli-Bacillus subtilis shuttle plasmid pBE2 A by restriction enzyme digestion,the recombinant plasmid pBE2A-BA2380 was constructed.After electroporating recombinant plasmid into A16D2?BA2380::spc,the reventant complementation mutant pBE2A-BA2380 / A16D2 was obtained.Finally,the growth characteristics and spore formation of BA2380 gene deleted mutant A16D2?BA2380::spc and the reventant complementation mutant pBE2A-BA2380 / A16D2 were studied using the starting strain A16D2 as the control.There were no significant difference in the growth and spore formation among the three strains,it was indicated that deleted BA2380 gene had no effect on the growth and spore formation of B.anthracis.The expression of BA2380 gene in A16D2,A16D2?BA2380::spc,pBE2A-BA2380 / A16D2 and pBE2 A / A16D2 was simultaneously analyzed by Western blot,BA2380 gene was expressed only in the starting and reventant complementation mutant.The results further demonstrated that BA2380 gene deletion and reventant complementation mutant were successfully constructed.This study will lay the foundation study of B.anthracis BA2380 gene function and its correlation with anthrax virulence and S-layer protein degradation.This work also provides scientific data for illuminating the pathogenicity of B.anthracis and the new vaccine research of B.anthracis for further.