卡氏棘阿米巴肌动蛋白1的免疫学特性和细胞黏附功能  

Immunological characteristics and cellular adhesion function of Acanthamoeba castellanii Actin 1

作　　者：李晶 杨舒越 赵佳欣 孔繁利[1] 郭思瑶 冯宪敏 LI Jing;YANG Shuyue;ZHAO Jiaxin;KONG Fanli;GUO Siyao;FENG Xianmin(Department of Pathophysiology,School of Basic Medical Sciences,Beihua University,Jilin 132013,China;Department of Pathogenic Biology,School of Basic Medical Sciences,Jilin Medical University,Jilin 132013,China)

机构地区：[1]北华大学基础医学院病理生理教研室,吉林吉林132013 [2]吉林医药学院基础医学院病原生物学教研室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2023年第2期308-314,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅自然科学基金项目(20220101307JC);吉林省卫健委卫生与健康技术重点创新项目(2020J012);吉林省教育厅科学技术重点研究项目(JJKH20210053KJ)。

摘　　要：目的:探讨卡氏棘阿米巴(Ac)肌动蛋白1 (Actin 1)(Ac-Actin 1)的免疫学特性,初步阐明Ac-Actin 1介导Ac虫体黏附宿主细胞并参与Ac虫体入侵的作用。方法:以Ac滋养体cDNA为模板,构建原核表达载体pET 22b (+)-Ac-Actin 1,转化大肠埃希菌感受态细胞BL21 (DE3);1 mmol·L^(-1)异丙基硫代半乳糖苷(IPTG)体外诱导表达重组Ac-Actin 1蛋白(rAc-Actin 1),十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对rAc-Actin 1进行可溶性分析,通过亲和层析方法纯化带有His标签的rAc-Actin 1。3只新西兰白兔随机分为对照组(1只)和实验组(2只);实验组兔以rAc-Actin 1为免疫原皮下注射400μg,对照组兔注入等量生理盐水,制备抗rAc-Actin 1多克隆兔血清;采用酶联免疫吸附试验(ELISA)法检测2组兔抗rAc-Actin 1多克隆抗体效价并测定IgG亚型,Western blotting法检测抗rAc-Actin 1多克隆兔血清与rAc-Actin 1的免疫反应性,间接免疫荧光实验(IFA)检测Ac-Actin 1在Ac滋养体中的定位。以正常滋养体为对照,抗rAc-Actin 1多克隆兔血清阻断Ac滋养体后与Vero细胞共孵育,显微镜观察Ac-Actin 1对Vero细胞的黏附作用。结果:SDSPAGE和BCA蛋白浓度检测,获得高浓度rAc-Actin 1 (1.7 g·L^(-1))蛋白。ELISA法检测,制备的兔抗rAc-Actin1特异性多克隆抗体效价为1∶6 400, IgG1和IgG2a浓度分别为116.76 g·L^(-1)和1 136.15 mg·L^(-1)。Western blotting法检测,兔抗rAc-Actin 1多克隆抗体可与rAc-Actin 1发生特异性结合,具有良好的免疫反应性。IFA检测,rAc-Actin 1主要定位于Ac滋养体的细胞膜上。显微镜观察,与对照组比较,随着抗体与虫体孵育时间的延长,实验组Ac滋养体对Vero细胞的黏附率明显降低(P<0.01),抗rAc-Actin 1特异性多克隆抗体可有效阻断Ac滋养体与Vero细胞的黏附。结论:rAc-Actin 1具有良好的免疫原性和免疫反应性,参与Ac虫体与宿主细胞的黏附作用。objective:To investigate the immunological characteristics of Acanthamoeba castellanii(Ac)Actin(Actin 1)(Ac-Action 1),and to clarify the role of Ac-Actin 1 in adhesion to the host cells and invasion of Ac parasite preliminarily.Methods:The Ac trophozoite cDNA was regarded as the template,and the prokaryotic expression vector pET 22b(+)-Ac-Actin 1 was constructed and transformed into the Escherichia coli competent cells BL21(DE3);the recombinant Ac-Actin 1 protein(rAc-Actin 1)was induced by 1 mmol·L^(-1) isopropylβ-D-thiogalactoside(IPTG)in vitro,and the solubility of rAc-Actin 1 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE);the rAc-Actin 1 with His label was purified by affinity chromatography.Three New Zealand white rabbits were randomly divided into control group(n=1)and experiment group(n=2);the rabbits in experiment group were subcutaneously injectied with 400μg rAc-Actin 1 as the immunogen and the rabbit in control group was injected with the same amount of normal saline,and the anti-rAc-Actin 1 polyclonal serum of the rabbits was prepared;the titers of anti-rAc-Actin 1 polyclonal antibody of the rabbits in two groups were determined by enzyme linked immunosorbent assay(ELISA)method and the IgG subtype was determined;Western blotting method was used to detect the immunoreactivity of anti-rAc-Actin 1 polyclonal rabbit serum and rAc-Actin 1;the localization of Ac-Actin 1 in the Ac trophozoite was detected by the indirect immunofluorescence assay(IFA).The normal trophozoites were used as controls,the anti-rAc-Actin 1 polyclonal serum of the rabbits blocked the Ac trophozoites and was co-incubated with the Vero cells,and the adhesion effect of Ac-Actin 1 on the Vero cells was observed by microscope.Results:The SDSPAGE and BAC concentration detection results showed that the high concentration of rAc-Actin 1(1.7 g·L^(-1))protein was obtained.The ELISA results showed that the titer of the obtained rabbit anti-rAc-Actin 1 specific polyclonal antibody was 1∶6400,and the con

关 键 词：卡氏棘阿米巴 滋养体 细胞黏附 免疫原性 免疫反应性 

分 类 号：R382.1[医药卫生—医学寄生虫学]