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

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

Key words:  Alpha–toxin  of Clostridium  welchii type  A, Site-directed  mutagenesis, Gene  expression, Phospholipase  C activities, Circular  dichroism spectra

摘要:

利用PCR定点突变技术,将A型魏氏梭菌α毒素的第56位天冬氨酸(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)。经酶切鉴定和序列分析证实,构建的表达质粒pM-D56G含有α毒素D56G突变基因。经SDS-PAGE分析,重组菌株BL21(DE3) (pM-D56G)中α毒素D56G突变体蛋白表达量占菌体总蛋白相对含量的22.48%。应用EXPASY服务器上的SOPMA法预测了α毒素和α毒素D56G突变体蛋白二级结构和同源模建其3D结构,结果两者的二级结构主要由α螺旋和无规则卷曲组成,两者的3D结构非常相似。圆二色(CD)光谱仪测定了α毒素和α毒素D56G突变体蛋白的CD光谱,结果两者的CD光谱有一些微小变化。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。免疫攻毒试验结果表明,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。

Abstract:

The site 56 (Asp-56→Gly-56,GAT→GGT) mutation which might influence the bioactivity of alpha-toxin of Clostridium welchii type A was induced by PCR site-directed mutagenesis technique. The recombinant plasmid pM-D56G containing alpha-toxin D56G codingmutant gene was obtained and transformed into Escherichia coli BL21(DE3) by recombimant DNA technique. The recombinant plasmid pM-D56G contained the alpha-toxin mutant gene which had expected mutation was confirmed by endonuclease-digestion and sequence analysis. The expression level of the alpha-toxin D56G mutant proteins of the recombinant strain BL21(DE3) (pM-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 alpha-toxin and alpha-toxin D56G mutant proteins were predicted by using SOPMA method on EXPASY website. The results showed that the secondary structure of alpha-toxin and alpha-toxin D56G mutant proteins was composed of alpha helices and random coils and their three-dimensional structure was very similar. 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 alpha-toxin. 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.