摘要
利用定点突变技术,将A型产气荚膜梭菌α毒素(CPA)第272位苏氨酸(ACA)定点突变成脯氨酸(CCG),构建了含CPA突变基因表达质粒的重组菌株BL21(DE3)(pMCPA-T272P)。经酶切鉴定和序列测定证实,构建的重组质粒pMCPA-T272P含有CPA-Thr272Pro突变基因,且基因序列和阅读框架正确。重组菌株BL21(DE3)(pMCPA-T272P)表达产物经SDS-PAGE分析,其表达量占菌体总蛋白相对含量的18.34%。应用ExPASy服务器上的SOPMA法对CPA和CPA-Thr272Pro突变体分子进行二级结构预测,同时模拟了其3D结构。结果显示,CPA和CPA-Thr272Pro突变体蛋白的二级结构主要是由α-螺旋和无规则卷曲组成,3D结构非常相似。CPA和CPA-Thr272Pro突变体蛋白的圆二色(CD)光谱分析发现二者的CD光谱有一些微小变化。磷脂酶C活性检测结果表明,CPA-Thr272Pro突变体蛋白失去了α毒素的磷脂酶C活性。免疫试验结果表明,用CPA-Thr272Pro突变体蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。
A site 272（Thr→Pro） mutation which might influence the bioactivity of alpha toxin of Clostridium perfringens was induced by site-directed mutagenesis technique.The recombinant plasmid pMCPA-T272P containing alpha toxin-coding mutant gene was obtained and transformed into Escherichia coli BL21（DE3）.The recombinant plasmid pMCPA-T272P which had expected mutation was confirmed by endonuclease-digestion and sequence analysis.The expressed product of the recombinant strain BL21（DE3）（pMCPA-T272P） was about 18.34% of total cellular proteins estimated in SDS-PAGE analysis.The induced secondary structure and three-dimensional structure（3D） of CPA and CPA-Thr272Pro mutant proteins were predicted by using SOPMA method on ExPASy website.The results showed that the secondary structure of CPA and CPA-Thr272Pro is composed of alpha helices and random coils and their three-dimensional structure was very similar.Circular Dichroism（CD） spectra of CPA and CPA-Thr272Pro proteins were a little difference by CD analysis.CPA-Thr272Pro mutant protein did not exhibit any phospholipase C（PLC） activity in bilogical activities of the toxin.Immunization in a mouse model with inactivated recombinant strain induced protection against at least 1MLD of the toxin from Clostridium perfringens type A.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第10期186-192,共7页
Biotechnology Bulletin
基金
国家自然科学基金项目(30972188)
关键词
产气荚膜梭菌
Α毒素基因
定点突变
基因表达
圆二色光谱
Clostridium perfringens Alpha-toxin gene Site-directed mutagenesis Gene expression Circular Dichroism（CD） spectra
