日本血吸虫组织蛋白酶L样基因的原核表达、纯化与活性分析  

Prokaryotic expression,purification and activity analysis of Schistosoma japonicum cathepsin L like gene

作　　者：陈奕慧[1] 李孜[2] 

机构地区：[1]广州医科大学附属第二医院医院感染管理科,广州510260 [2]广州医科大学病原生物学与免疫学教研室,广州510182

出　　处：《国际医学寄生虫病杂志》2015年第5期249-254,共6页International JOurnal of Medical Parasitic Diseases

基　　金：国家自然科学基金(30600516);广州市科技计划(2012J4100009)~~

摘　　要：目的：在原核系统中表达日本血吸虫组织蛋白酶L样（Schistosoma japonicum cathepsin L， SjCLs）基因，并检测所表达SjCLs重组蛋白的生物学活性。方法将SjCLs基因克隆入原核表达载体pET-30a （＋），并转化大肠埃希菌 BL21（DE3），用异丙基-β-D-硫代半乳糖苷（isopropyl-β-D-thio-galactoside，IPTG）诱导重组蛋白表达，用亲和层析柱纯化表达产物，对表达的重组蛋白及纯化产物进行聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，采用蛋白印迹（Western blot）分析日本血吸虫感染兔血清与SjCLs 重组蛋白之间免疫反应性。以N-苄酯基-苯丙氨酸-精氨酸7-酰胺基-4-甲基香豆素（Z-Phe-Arg-Nmec）为底物，采用荧光分光光度计分析重组蛋白的酶活性。结果成功构建pET30a-SjCLs重组质粒，并在大肠埃希菌中表达获得SjCLs重组融合蛋白，SDS-PAGE显示，表达及纯化蛋白的相对分子质量约为38000，与SjCLs的理论相对分子质量基本一致。SjCLs重组蛋白的免疫反应性分析结果为阴性，表明重组蛋白不能被抗日本血吸虫感染兔血清识别。以Z-Phe-Arg-Nmec为反应底物，检测到SjCLs的吸光度（A460）值为925，证实SjCLs具有组织蛋白酶的活性。结论获得了原核表达的SjCLs重组蛋白，该重组蛋白具有类似于SjCL2的酶活性，为深入研究SjCLs的功能奠定了基础。Objective To express the Schistosoma japonicum cathepsin L like （SjCLs） gene in prokary-otic expression system and assay the biological activity of the SjCL recombination protein. Methods The SjCLs gene was subcloned into the pET30a （＋） vector to generate the expression recombinant construct：pET30a （＋）-SjCLs. This construct was then transformed into E. coli BL21 （DE3） to obtain the recombinant SjCLs protein with the induction of IPTG. The recombinant proteins were purified by affinity chromatography. The purified proteins were then analyzed with SDS-PAGE and Western blot. The enzyme activity of the fusion protein was determined by fluorospectrophotometer using Z-Phe-Arg-Nmec as a cathepsin substrate. Results SjCLs fusion proteins were obtained in the transformed E. coli cells by IPTG induction. SDS-PAGE analysis using purified proteins showed a protein band locating at about Mr 38 000, consistent with the predicted molecular weight of the fusion protein. Western blot analysis showed that this recombinant protein could not be recognized by the serum of S. japonicum-infected rabbit. Using Z-Phe-Arg-Nmec as a substrate for the re-combinant proteins,the absorbance value of reaction system was 925 at 460 nm, suggesting an enzymatic ac-tivity of the SjCLs proteins. Conclusions The SjCLs recombinant protein was obtained using prokaryotic expression system. This protein has an enzymatic activity similar to that of SjCL2 which will help to furtherinvestigate the function of SjCLs.

关 键 词：日本血吸虫 组织蛋白酶 原核表达 重组蛋白 

分 类 号：R532.21[医药卫生—内科学]