十二指肠钩虫半胱氨酸蛋白酶抑制剂的分离、表达与活性研究  

Isolation, expression and inhibitory activity of cysteine protease inhibitors (cystatins) of Ancylostoma duodenale

作　　者：邵正 邓莉 崔咏诗 卢晓湧 张菊英 卢玲玲 许琴英 陈传 何庆丰 彭礼飞 SHAO Zheng;DENG Li;CUI Yong-shi;LU Xiao-yong;ZHANG Ju-ying;LU Ling-ling;XU Qin-ying;CHEN Chuan;HE Qing-feng;PENG Li-fei(Department of Parasitology,Guangdong Medical University,Zhanjiang 524023,Guangdong,China;School of Medical Technology,Guangdong Medical University)

机构地区：[1]广东医科大学寄生虫学教研室,广东湛江524023 [2]广东医科大学医学技术学院

出　　处：《中国病原生物学杂志》2022年第11期1278-1282,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31101639);广东省高等学校特色创新科研项目(No.2015KTSCX050);广东医科大学科技创新团队项目(No.STIF201107)。

摘　　要：目的分离、克隆十二指肠钩虫半胱氨酸蛋白酶抑制剂AduCPI1-AduCPI4基因,在大肠埃希菌中进行表达,了解重组AduCPI1-AduCPI4对组织蛋白酶(cathepsin,cat)的抑制作用。方法根据预测的4种编码AduCPI基因序列(GenBank No JQ762416-JQ762419)设计引物,采用PCR技术从十二指肠钩虫成虫cDNA中扩增AduCPIs成熟蛋白编码基因,并克隆、连接到表达质粒pET32a-sumo,构建重组原核表达载体。重组载体转入到E.coli BL21(DE3),用IPTG诱导表达融合蛋白。通过Ni-NTA亲和层析纯化破碎菌上清中的重组融合蛋白,在纯化柱上用SUMO蛋白酶酶切融合伴侣,收集穿透液获得纯化的重组目的蛋白,采用SDS-PAGE分析蛋白表达及纯化情况,采用酶活性抑制试验检测4种重组AduCPI对catB、catL和catS的抑制作用。结果扩增并克隆获得了4种AduCPI的成熟蛋白编码基因,构建的重组表达载体转化大肠埃希菌后表达相应蛋白,经纯化获得重组AduCPI1-AduCPI4。4种AduCPI抑制catS(0.5 nmol/L)(pH 6.5或7.4)的IC_(50)在(0.60±1.13)nmol/L~(2.89±1.37)nmol/L,抑制catL(0.5 nmol/L)(pH 5.5)的IC_(50)在(0.50±1.06)nmol/L~(1.42±1.07)nmol/L,抑制catB(0.5 nmol/L)(pH 6.0或7.4)的IC_(50)在(5.24±1.03)nmol/L~(68.90±1.38)nmol/L,其中以AduCPI4对3种组织蛋白酶的抑制作用最强,而AduCPI2对catS、catB(pH7.4)的IC_(50)分别为(0.80±1.08)nmol/L和(68.90±1.38)nmol/L,抑制作用效率差异较大,AduCPI2对catS具有较好的选择性抑制作用。结论克隆表达了4种AduCPI,该4种蛋白对组织蛋白酶L和S均具有很强的抑制作用,对组织蛋白酶B的抑制作用也较强,研究结果为进一步了解AduCPIs的生物学功能及其应用奠定了基础。Objective To isolate,clone and overexpress AduCPI1-AduCPI4,the cysteine protease inhibitors of Ancylostoma duodenale,and detect their inhibitory activity on cathepsin B(catB),L(catL)and S(catS).Methods The primers were designed according to the predicted gene sequences encoding four AduCPIs(GenBank No JQ762416-JQ762419),and the nucleotide sequences encoding AduCPI1-AduCPI4 mature protein were PCR amplified from the A.duodenale cDNA,then ligated into pET32 a-sumo to construct the recombinant plasmids,respectivily.The 4 recombinant fusion proteins were inductively expressed in Escherichia coli BL21(DE3)by IPTG,and purified by Ni-NTA affinity chromatography,respectively.The purified rAduCPI was obtained after cleavage of their fusion tag with SUMO protease on the resin bed.The protein expression and purification were analyzed by SDS-PAGE,and the inhibitory effects of rAduCPIs on catB,catL and catS were detected by microplate reader.Results The gene encoding 4 AduCPIs mature protein was amplified and cloned from the A.duodenale cDNA,respectively,and each rAduCPI was successfully expressed and purified in E.coli.These four AduCPIs inhibited catS(0.5 nmol/L)(pH 6.5 or 7.4)with IC_(50) of(0.60±1.13)nmol/L-(2.89±1.37)nmol/L,inhibited catL(0.5 nmol/L)(pH 5.5)with IC_(50) of(0.50±1.06)nmol/L-(1.42±1.07)nmol/L,and inhibited catB(0.5 nmol/L)(pH 6.0 or 7.4)with IC_(50) of(5.24±1.03)nmol/L-(68.90±1.38)nmol/L,of which AduCPI4 was the most effective inhibitor in inhibiting cathepsin B,L and S,while the IC_(50) of AduCPI2 on catS and catB(pH7.4)was(0.80±1.08)nmol/L and(68.90±1.38)nmol/L,respectively,which showed differences in inhibition efficiency,and AduCPI2 had a selective inhibitory effect on catS.Conclusion Four AduCPIs were cloned and expressed in this study,and all these AduCPIs are highly potent inhibitors of cathepsin L and S,and strong inhibitors of cathepsin B.The results of this study lay the foundation for further understanding of the biological function of AduCPIs and their application.

关 键 词：十二指肠钩虫 半胱氨酸蛋白酶抑制剂 原核表达 组织蛋白酶 

分 类 号：R383.13[医药卫生—医学寄生虫学]