Screening Of Anti-phage And Anti-bacterialgenes By Using Ba Cillus Subtilis Expression System

Bacillus subtilis is a gram positive bacterium,which is a safe expression system to the environment.It is also charactered as a non pathogenic,easy cultivation,grow fast,genetic background clear,strong ability of protein secretion,and it has great potential and advantages as the genetic engineering bacteria.It is widely applied to various fields.In order to reduce extracellular protease degradation of heterologous protein,mutation of the eight key extracellular proteases of B.subtilis WB800 strain was taken as the expression strain.At the same time,the signal peptide library was integrated into the shuttle expression vector pBE-S of B.subtilis by the homologous recombination principle.The processed cDNA of Pinellia ternata and expression vector pBE-S with signal peptide library were randomly fused to construct a cDNA Library.A total of 1772 monoclonal transformants were obtained.Isolation and identification of the phage of B.subtilis WB800 are important to screen resistant genes against phage.A B.subtilis phage was successfully isolated in this study.Electron microscopic observation showed that the head of this phage particle is a hexahedron,and its size is about 43nmX33nm.It has a short tail,and the length of the tail is about 40nm.After phage DNA extraction,digestion,vector ligation,and sequencing,DNA sequence analysis proved that it is a B.subtilis phage Nf.Screening the resistant genes against phage from the cDNA library,several methods were explored.The study on screening of antimicrobial gene found that some transformants appeared autolysis.By using this method,42 apparent autolysis transformants were obtained from the cDNA library of P.ternata.These 42 clones were sequenced and analyzed..BLAST analysis found that all the sequences were not similar to any gene from the NCBI database.Among the 42 clones,15 plasmid genes were isolated and retransforred to B.Subtilisto determine the stability of the autolysis.Twelve genes were confirmed that can cause autolysis again after retransformation.Crude protein from 1168 strain of B.Subtilis was proved that it has obvious inhibiting effect on B.subtilis itself.Tricine-SDS-PAGE of the crude protein of 1168 strain showed that there was a specific band approximately 8kd,and its size matched with predicted protein.This study explored an effective method to screen the antibacterial genes,which is a good preparation for the following research.