Expression And Characteristic Identification Of Asp-56-site-directed Mutant And Amino-terminal Domain Plc Gene Of Alpha-toxin From Clostridium Welchii Type A

The alpha-toxin(Clostridium welchii alpha-toxin, α-toxin) is a major pathogenic factor of Clostridium welchii type A which can cause traumatic gas gangrene on human or livestock and human food poisoning. The α-toxin is a metalloenzyme and exhities phospholipase C(PLC) activity. The α-toxin possessees enzymic molecular struture feature with catalytic site and binding site. Some amino acid residues of the α-toxin are important parts of catalytic and active groups, which also play an important role in the formation of its biological activity. The α-toxin amino-terminal domain possesses PLC activity. It is unknown the relationship between molecular struture and function of α-toxin. This study is to research the site 56(Dâ†'G) mutation which might influence the bioactivity of α-toxin, gene expression and structural analysis and its characteristic of α-toxin amino-terminal domain of Clostridium welchii type A.On the one hand, the site 56(Asp-56â†'Gly-56,GATâ†'GGT) mutation which might influence the bioactivity of α-toxin of Clostridium welchii type A was induced by PCR site-directed mutagenesis technique. The recombinant plasmid p M-D56 G containing α-toxin D56 G coding mutant gene was obtained and transformed into Escherichia coli BL21(DE3) by recombimant DNA technique. The recombinant plasmid p M-D56 G contained the α-toxin mutant gene which had expected mutation was confirmed by endonuclease-digestion and sequence analysis. The expression level of the α-toxin D56 G mutant proteins of the recombinant strain BL21(DE3)(p M-D56G) was about 22.48% of total cellular proteins with IPTG indution by SDS–PAGE analysis. The deduced secondary structure and three-dimensional structure(3D) of α-toxin and α-toxin D56 G mutant proteins were predicted by using SOPMA method on EXPASY website. The results showed that the secondary structure of α-toxin and alpha-toxin D56 G mutant proteins was composed of alpha helices and random coils and their three-dimensional structure was very similar. The circular dichroism(CD) spectra of α-toxin D56 G mutant proteins and α-toxin had some small changes by CD analysis. The biological activity results indicated that the alpha-toxin D56 G mutant proteins were loss of phospholipase C activity of α-toxin. More importantly, immunization in a mouse model with crude preparation containing the α-toxin D56 G protein inclusion bodies induced protection against at least 1MLD of the α-toxin of Clostridium welchii type A.On the other hand, amino-terminal domain 1-250 residues gene of Clostridium welchii α-toxin(α-toxin PLC1-250) was amplified by PCR. The recombinant plasmid p N-PLC containing plc gene was obtained and transformed into Escherichia coli BL21(DE3) by recombimant DNA technique. The recombinant plasmid p N-PLC contained the α-toxin amino-terminal domain 1-250 residues gene was confirmed by endonuclease-digestion and sequence analysis. The expression level of the α-toxin PLC1-250 proteins was about 18.76% of total cellular proteins with IPTG indution by SDS–PAGE analysis. The deduced secondary structure and three-dimensional structure(3D) of α-toxin and α-toxin PLC1-250 proteins were predicted by using SOPMA method on EXPASY website. The results showed that the secondary structure of α-toxin and α-toxin PLC1-250 proteins was composed of alpha helices and random coils. The three-dimensional structure of α-toxin PLC1-250 protein was similar with amino-terminal domain of α-toxin protein. The circular Dichroism(CD) spectra of α-toxin and α-toxin PLC1-250 proteins were similar by CD analysis. The biological activity results indicated that the α-toxin PLC1-250 proteins still possess phospholipase C(PLC) activity of α-toxin.In a word, this study provide theory basis and experimental data to study genetically engineered subunit vaccine and molecular mechism of α-toxin of Clostridium welchii type A. It is important theory value to elucidate the pathogenic mechism of α-toxin of Clostridium welchii type A.