广州管圆线虫半胱氨酸蛋白酶抑制剂基因的克隆表达及免疫保护作用研究  

Cloning,expression and immunoprotection efficacy of the cystatin gene of Angiostrongylus cantonensis

作　　者：潘智华[1,2] 何蔼[1] 程梅[1] 李卓雅[1] 于彦杰[1] 贺华良[1] 詹希美[1] 

机构地区：[1]中山大学中山医学院寄生虫学教研室,广州510089 [2]徐州医学院形态学实验室,徐州221004

出　　处：《中国人兽共患病学报》2009年第6期534-538,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金-广东省人民政府联合基金(U0632003)资助项目

摘　　要：目的对广州管圆线虫半胱氨酸蛋白酶抑制剂(cystatin)基因进行克隆、原核表达,并对表达产物的免疫保护作用作出初步鉴定。方法将广州管圆线虫cystatin基因克隆入载体pGEX-4T-1中,转化大肠杆菌BL21进行诱导表达。用纯化融合蛋白隔周免疫小鼠1次,共3次,设GST免疫及未免疫对照组,末次免疫后1w用广州管圆线虫Ⅲ期幼虫攻击感染,3w后剖杀小鼠,计算减虫率及保护率,并测定小鼠特异性IgG抗体水平变化。结果成功构建了pGEX-4T-1/cystatin重组表达质粒,并在大肠杆菌中得以高效可溶性表达,融合蛋白免疫小鼠后诱导产生了58.1%的减虫率及20%的保护率,与对照组相比减虫效果显著(P<0.01)。结论广州管圆线虫cystatin基因能在大肠杆菌中高效表达并可诱导小鼠产生一定水平的保护性免疫力。To clone and express the cystatin gene of Agiostrongylus cantonensis （AC） and to identify the immunoprotecrive effect of the recombinant protein, the cystatin cDNA was subcloned into the expression vector pGEX-4T-1. Then the recombinant vector was transferred into E. coli BL21（DE3） and expressed. Mice were immunized 3 times in every week interval with the recombinant protein, and the control mice were immunized with GST protein. One week after the final immunization, all mice were challenged with the third-stage larvae of A. cantonensis. All mice were killed to count the larvae reduction rate and protection rate 3 weeks after challenge. Besides, serum levels of the cystatin-specific antibody （IgG） in mice were detected. The recombinant plasmid pGEX-4T-1-ACcystatin was constructed successfully and the fusion protein was highly expressed in E. coli in soluble form. The number of larvae of A. cantonensis were evidently reduced in mice immunized with fusion protein（P 〈0.01）. The larvae reduction rate and protection rate were 58.1 % and 20% respectively. It is evident that the cystatin gene of A. cantonensis could be high expressed in E. coli and the fusion protein can induce protective immunity in mice.

关 键 词：广州管圆线虫 半胱氨酸蛋白酶抑制剂 分子克隆 免疫保护 

分 类 号：R383.1[医药卫生—医学寄生虫学]