Comparative Identification Of Two Bacteria And Expression Of Non-hemolytic Enterotoxin Genes

The degree of bacterial contamination in the surrounding environment is an important issue in the healthcare industry.In this study,two strains of bacteria isolated from a hospital were selected as the research object.These two kinds of bacteria were identified and the proteomic differences between the two strains were compared.The main contents and conclusions of this dissertation are as follows:(1)Two strains of bacteria were identifiedThe phylogenetic tree analysis was constructed based on phenotypic,physiological and biochemical characteristic responses and 16S rRNA gene sequences.The results showed that the bacteria 65-5 was Bacillus subtilis,and the bacteria 41-1 was Bacillus cereus sensu lato.(2)The comparative analysis of proteomic between two strains of bacteriaThe proteomic differences between the two bacteria were compared by two-dimensional electrophoresis tandem mass spectrometry.There were significant differences in protein spots between the two bacterial proteins.Through the identification of mass spectrometry,the strong pathogenic bacteria 41-1 was further determined the Bacillus cereus.By studying the proteomics of protein secretion by Bacillus cereus,it was found that proteins from the supernatant of the Bacillus cereus contain flagellin and metalloproteinases.(3)PCR identification of pathogenic genes in Bacillus cereusThe 41-1 strain was identified as Bacillus cereus by strain identification.Bacillus cereus is a conditioned pathogen,which can produce a variety of pathogenic toxins.The toxin gene of Bacillus cereus was identified by PCR.Results showed that the strain contained the non-hemolytic enterotoxin gene nhe A,nhe B,nhe C,but not hemolytic enterotoxin gene hbl A,hbl C,hbl D,cytotoxin K gene cyt K or enterotoxin Cereulide gene ces.Additionally,the cloning of three pathogenic gene nhe A,nhe B,nhe C were obtained.(4)Prokaryotic expression of three genes nhe A,nhe B,nhe CUsing these three cloning pathogenic genes nheA,nheB,nheC genes,pET-30a(+)-nhe A,pET-30a(+)-nhe B and pET-30a(+)-nhe C expression vectors were constructed into Escherichia coli.Three non-hemolytic enterotoxin subunits were successfully expressed in Escherichia coli.Nhe B subunits are prone to form polymers when expressed in prokaryotic cells.The mass spectrometry verify that three proteins were expressed correctly.The three non-hemolytic enterotoxin proteins purified by tapping were used as antigens to immunize New Zealand white rabbits to obtain the polyclonal antiserum of the three target proteins.The effect of polyclonal antibody was identified by prokaryotic expression of the corresponding recombinant protein.Results show that three polyclonal antibodies were successfully produced.The protein in the supernatant of Bacillus cereus was tested with the prepared polyclonal antibody.Non-hemolytic enterotoxin subunits Nhe A and Nhe B were specifically identified while non-hemolytic enterotoxin Nhe C subunits were not identified.(5)Construction of shuttle plasmidsThe relationship between the function and interaction of three non-hemolytic enterotoxins was studied.Three non-hemolytic enterotoxin genes were cloned into the shuttle vector of baculovirus using the AcMNPV bacmid expression system,and the corresponding shuttle plasmid was obtained.The shuttle plasmid was transformed into Bacmid competent cells for transposition.Plasmid pACnhe C-nhe A-nhe B bacmid,pACnhe c-nhe A bacmid,pACnhe C-nhe B bacmid were successfully constructed.