A型魏氏梭菌α毒素Asp-56基因定点突变与结构分析  

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

作　　者：宫语晨 许崇利[2] 钱爱东[1] 许崇波[3] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]吉林化工学院化工与生物技术学院,吉林吉林132022 [3]大连大学医学院,辽宁大连116622

出　　处：《中国兽药杂志》2014年第6期1-7,共7页Chinese Journal of Veterinary Drug

基　　金：国家自然科学基金项目(30972188)

摘　　要：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。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.

关 键 词：A型魏氏梭菌 Α毒素 基因定点突变 磷脂酶C活性 圆二色光谱 

分 类 号：S852.61[农业科学—基础兽医学]