多房棘球蚴抗原亮氨酸氨基肽酶的原核表达纯化及酶活性分析

Purification of prokaryotic expression and analysis of enzyme activityof Leucine Aminopeptidase(LAP)in Echinococcus Multilocularis

目的构建多房棘球坳抗原亮氨酸氨基肽酶(Leucine aminopeptidase,LAP)原核表达系统,并做纯化及最佳酶活性条件分析,为后续作用机制研究奠定基础。方法利用Optimum^TM Codon软件对LAP编码序列进行优化后合成目的基因,连接入表达载体pCzn1后转化入大肠杆菌BL-21菌株,在发酵过程中加入IPTG诱导目的蛋白表达后,通过Ni柱亲和纯化获得高纯度重组目的蛋白LAP。以亮氨酸对硝基苯胺(Leu-pNA)为底物测定LAP酶活性,观察酶活性的最适pH和温度,以及不同金属离子对其酶活性的影响。结果﹐基因测序和酶切鉴定显示pCzn1-LAP质粒构建成功,通过Ni柱亲和纯化得到一个大小约为57KD的蛋白。当pH为9.0、温度为60℃时LAP酶活性最强;金属离子对LAP酶活性影响的小大顺序为Co^2+>Zn^2+>Ca^2+>Mn^2+>Mg^2+>Ba>K^+。结论本研究成功构建LAP原核表达系统,表达和纯化了LAP蛋白,获得了酶活性最强时的基础条件,为研发抗寄生虫药物及探究LAP在多房棘球坳感染过程的作用机制奠定了基础。

Objective To construct a prokaryotic expression system of Leucine Aminopeptidase(LAP)and to havepurification as well as conditional analysis of the optimal enzyme activity so as to lay a foundation for further study on theactive mechanism.Methods After optimizing the coding sequence of LAP by using Optimum TM Codon,the target gene was synthesized and inserted into the expression vector pCanl to prepare BL-21 strains of colibacillus.After IPTG was addedinto the fermentation process to induce the expression of the target protein,LAP of high-purity was obtained by means ofthe affinity purification of Ni column.The enzyme activity of LAP,the optimal pH and temperature of enzyme activit as well as the effects of dfferent metal ions on the enzyme activity were determined and observed based on Leu-pNA.Re-sults The pCan1-LAP plasmid was successfully constructed by gene sequencing and enzyme digestion.A protein with asize of 57KD was obtained by the affinity purification of Ni column.The enzyme activity of LAP was the strongest whenpH is 9.0 and temperature is 60℃.The order of Em-LAP activity in different metal ion chloride solutions was Co^2+>Zn^2+>Ca^2+>Mn^2+>Mg^2+>Ba>K^+.Conclusion This study successfully constructs a prokaryotic expression system of LAP,ex-pressts and purifies LAP protein as well as obtains the basic conditions for the strongest enzyme activity,which lays afoundation for the development of antiparasitic drugs and the active mechanism in the infection process of Echinococcusmultilocularis.