产气荚膜梭菌α毒素基因的定点突变与表达  

Site-directed Mutagenesis and Expression of Clostridium perfringens Alpha-toxin Gene

作　　者：许崇利[1,2] 许崇波[2] 

机构地区：[1]吉林化工学院环境与生物工程学院,吉林吉林132022 [2]大连大学医学院,辽宁大连116622

出　　处：《江苏农业学报》2010年第2期258-263,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(30972188)

摘　　要：选择可能影响α毒素生物活性的氨基酸相关碱基位点进行定点突变,构建α毒素基因的突变体并在大肠杆菌中表达。利用PCR定点突变技术,进行第68位组氨酸→亮氨酸的定点突变,构建含α毒素突变基因表达质粒的重组菌株BL21(DE3)pLys(pXMCPA02)。结果表明,α毒素突变基因第287位核苷酸由A→T,经酶切鉴定和序列测定证实,构建的重组质粒pXMCPA02含有α毒素突变基因,且基因序列和阅读框架正确;重组菌株BL21(DE3)pLys(pXMCPA02)表达产物经SDS-PAGE分析,其表达量占菌体总蛋白相对含量的33.82%。因此,已成功构建了α毒素基因突变体,并实现了在重组大肠杆菌中的表达。This study was to induce the mutation at the base site of amino acid which might influence the bioactivity of alpha-toxin,and to construct the mutant of alpha-toxin gene which could be expressed in Escherichia coli.A mutation at site 68（His→Leu） was induced by PCR site-directed mutation technique to construct the recombinant plasmid pXMCPA02 containing alpha-toxin mutant gene.The plasmid was then transformed into Escherichia coli BL21（DE3） pLys.The results showed that the nucleotide at site 287 of alpha-toxin mutant gene changed from A to T.The recombinant plasmid pXMCPA02 was identified containing the alpha-toxin mutant gene and having correct sequence and open reading frame（ORF） by endonuclease-digestion and sequence analysis.The expressed products of the recombinant strain BL21（DE3）pLys（pXMCPA02） were about 33.82% of total cellular protein by SDS-PAGE analysis.Taken together,the mutant of alpha-toxin was successfully constructed and expressed in Escherichia coli.

关 键 词：产气荚膜梭菌 Α毒素基因 定点突变 表达 

分 类 号：Q786[生物学—分子生物学]