A型魏氏梭菌α毒素Asp-56基因定点突变与结构分析

为探索A型魏氏梭菌α毒素第56位天冬氨酸(Asp-56)对其生物学活性的影响,将α毒素Asp-56密码子(GAT)定点突变成甘氨酸(Gly-56)密码子(GGT),构建了含α毒素D56G突变基因表达质粒的重组菌株BL21(DE3)(pM-D56G),该突变体蛋白表达量约为22.48%。α毒素和α毒素D56G突变体蛋白的二级结构主要由α螺旋和无规则卷曲组成,两者的圆二色(CD)光谱检测有微小变化。生物学活性检测结果表明,α毒素D56G突变体蛋白失去了α毒素的磷脂酶C活性,且α毒素D56G突变体蛋白免疫的小鼠能够保护1MLD的A型魏氏梭菌标准株C57-1毒素攻击。此研究为进一步研究α毒素分子结构与生物学功能的关系奠定了基础。

Site-directed Mutagenesis and Structure Analysis of Alpha-toxin Gene Asp-56 from Clostridium welchii Type A

To explore the biological activity of alpha-toxin Asp-56 from Clostridium welchii type A, the site 56 （Asp-56→Gly-56, GAT→GGT） mutation was induced by PCR site-directed mutagenesis technique. The recombinant strain BL21 （DE3）（pM-D56G） containing alpha-toxin D56G coding mutant gene was obtained by recombimant DNA technique. The expression level of the alpha-toxin D56G mutant proteins was about 22.48% of total cellular proteins. The deduced secondary structure of alpha-toxin and alpha-toxin D56G mutant proteins was composed of alpha helices and random coils. The circular dichroism （CD） spectra of alpha-toxin D56G mutant proteins and alpha-toxin had some small changes by CD analysis. The biological activity results indicated that the alpha-toxin D56G mutant proteins were loss of phospholipase C activity of alphatoxin. More importantly, immunization in a mouse model with crude preparation containing the alpha-toxin D56G protein inclusion bodies induced protection against at least 1MLD of the alpha-toxin of Clostridium welchii type A. This study provided a fundation for the relationship between the molecular struture and biological function of alphatoxin from Clostridium welchii type A.