1、第 49 卷 第 2 期2023年 3 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.49 No.2Mar.2023DOI：10.13481/j.1671587X.20230206卡氏棘阿米巴肌动蛋白 1的免疫学特性和细胞黏附功能李晶1,杨舒越1,赵佳欣1,孔繁利1,郭思瑶2,冯宪敏2（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）摘要 目的目的：探讨卡氏棘阿米巴（Ac）肌动蛋白 1（Actin 1）（Ac-Actin 1）的免疫学
2、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 L1 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-
3、Actin 1 为免疫原皮下注射 400 g，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
4、果结果：SDS-PAGE 和 BCA 蛋白浓度检测，获得高浓度 rAc-Actin 1（1.7 g L1）蛋白。ELISA 法检测，制备的兔抗 rAc-Actin 1 特 异 性 多 克 隆 抗 体 效 价 为 1 6 400，IgG1 和 IgG2a 浓 度 分 别 为 116.76 gL 1和1 136.15 mg L1。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，
5、实验组 Ac 滋养体对 Vero 细胞的黏附率明显降低（P0.01），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。结结论论：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。关键词 卡氏棘阿米巴；滋养体；细胞黏附；免疫原性；免疫反应性中图分类号 R382.1文献标志码 AImmunological characteristics and cellular adhesion function of Acanthamoeba castellanii Actin 1LI Jing1,YANG Shuyue1,ZHAO Ji
6、axin1,KONG Fanli1,GUO Siyao2,FENG Xianmin2（1.Department of Pathophysiology，School of Basic Medical Sciences，Beihua University，Jilin 132013，China；2.Department of Pathogenic Biology，School of Basic Medical Sciences，Jilin Medical University，Jilin 132013，China）文章编号 1671587X(2023)02030807收稿日期 20220507基金项
7、目 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）作者简介 李 晶（1995），女，吉林省吉林市人，检验技师，医学硕士，主要从事病原体分子致病机制方面的研究。通信作者 孔繁利，教授，硕士研究生导师（E-mail：）；冯宪敏，教授，博士研究生导师（E-mail：）308李晶，等.卡氏棘阿米巴肌动蛋白 1的免疫学特性和细胞黏附功能ABSTRACT objective：To investigate th
8、e 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
9、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 L1 isopropyl-D-thiogalactoside（IPTG）in vitro，and the solubility of rAc-Actin 1 was analyzed by sodium dodecyl sulfate-polyacr
10、ylamide 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 400 g rAc-Actin 1 as
11、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（
12、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 troph
13、ozoites 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 SDS-PAGE and BAC concentration detection results show
14、ed that the high concentration of rAc-Actin 1（1.7 g L1）protein was obtained.The ELISA results showed that the titer of the obtained rabbit anti-rAc-Actin 1 specific polyclonal antibody was 1 6 400，and the concentrations of IgG1 and IgG2a were 116.76 g L1 and 1 136.15 mg L1，respectively.The Western b
15、lotting results showed that the rabbit anti-rAc-Actin 1 polyclonal antibody could specifically bind to the rAc-Actin 1 and had good immunoreactivity.The IFA results showed that rAc-Actin 1 mainly located on the cell membrane of Ac trophozoites.The microscope analysis results showed that compared wit
16、h control group，the adhesion rate of Ac trophozoites to the Vero cells in the experiment group was significantly decreased（P0.01）with the prolongation of incubation time of antibody and parasite，and the anti-rAc-Actin 1 specific polyclonal antibody could effectively block the adhesion of Ac trophozoites to the Vero cells.Conclusion：rAc-Actin 1 has good immunogenicity and immunoreactivities，which is involved in the adhesion between the Ac parasite and the host cells.KEYWORDS Acanthamoeba castella
