不同表达条件对Eimeria tenella丙二酰单酰辅酶A:ACP转移酶活性的影响  

Effects of Different Expression Vectors and Temperatures on the Activity of Eimeria tenella Malonyl-CoA:ACP Transacylase

作　　者：孙铭飞[1] 覃宗华[1] 谢明权[1] 袁建丰[1] 吕敏娜[1] 余劲术[1] 吴彩艳[1] 蔡建平[1] 

机构地区：[1]广东省农业科学院兽医研究所广东省兽医公共卫生公共实验室,广州510640

出　　处：《畜牧兽医学报》2010年第9期1158-1165,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30471300);广东省国际科技合作专项(2008A050200015);广东省农业科学院重点基础研究项目(07-基础-07)资助

摘　　要：为探讨不同原核表达载体、不同表达温度对重组表达Eimeria tenella丙二酸单酰辅酶A:ACP转移酶活性的影响,以生物信息学技术预测的E.tenellaⅡ型脂肪酸合成代谢途径中的丙二酰单酰辅酶A:ACP转移酶(Et-MCAT)编码序列为基础,分别设计引物扩增完整ORF,采取截除信号肽而保留转导肽以及切除信号肽和转导肽的不同策略,选择pMAL-c2x、pET-32a(+)和pProExHTa3种不同表达载体分别构建重组表达质粒,转化大肠杆菌Rosseta(DE3)后,在不同条件下诱导表达,利用酮戊二酸脱氢酶(KDH)偶联系统测定表达产物的酶活性,以评价不同表达策略对表达效果和表达产物活性的影响。表达水平和酶活性分析结果显示,惟切除信号肽和转导肽后的功能蛋白编码区能表达,Rosseta(DE3)/pMAL-c2x-mcat体系的可溶性表达量高于Rosseta(DE3)/pET-32a(+)-mcat,但二者表达产物的酶活性相似,且均随着诱导温度的降低其可溶性蛋白表达量增加。但Rosseta(DE3)/pProExHTa-mcat体系仅能表达不溶性蛋白,且复性后无酶活性,改变诱导温度对表达效果无明显影响。结果表明不同表达载体和诱导温度对大肠杆菌所表达EtMCAT的活性及其表达形式有显著影响,其中融合标签和表达温度可能是主要的影响因素,包涵体表达的重组酶即使复性处理仍难达到酶活性分析的要求。This experiment was conducted to study the effects of different expression vectors and temperatures on the activity of recombinant Eimeria tenella Malonyl-CoA：ACP Transacylase （rEtMCAT）.Using the ORF sequence encoding EtMCAT predicted by bioinformatic analysis as a template,3pairs of primers were designed in order to amplify the following fragments：the entire ORF,the ORF without N-terminal signal peptide,the ORF without N-terminal signal peptide and transit peptide,respectively.Each of the 3cloned fragments was individually inserted into 3plasmid vectors pMAL-c2x,pET-32a（＋）and pProExHTa,respectively.All the recombinant vectors were transfected into E.coli Rosseta（DE3）,and induced with IPTG under different temperature conditions（37,30and 16℃）,respectively.The enzymatic activities of rEtMCAT were measured using the a-ketoglutarate dehydrogenase（KDH）-coupled assay system.The results revealed that only the ORF encoding the mature peptide,that is,excised the N-terminal signal peptide and transit peptide could be expressed in 3vector systems,and high content of soluble rEtMCAT was only expressed in recombinant plasmid pMAL-c2x-mcat or pET-32a（＋）-mcat,in which their expressed products had a similar enzymatic activity.The lower culture temperature （at 30and 16℃）can enhance their expression.But there was no any soluble protein expressed in Rosseta（DE3）/pProExHTa-mcat,and the recombinant protein expressed in inclusion body was no enzymatic activity even after renaturation.The characterizations of expression vectors used in experiments,especially the type of tag protein,as well as the expression temperatures have significant effects on the production and enzymatic activity of rEtMCAT expressed in E.coli Rosseta（DE3）.It is suggest that the rEtMCAT expressed in inclusion body can not be used for the enzymatic assay.

关 键 词：柔嫩艾美耳球虫 丙二酰单酰辅酶A:ACP转移酶 表达载体 酶活性 

分 类 号：S852.723[农业科学—基础兽医学] Q786[农业科学—兽医学]