卡氏棘阿米巴肌动蛋白1的免疫学特性和细胞黏附功能

doi:10.13481/j.1671-587X.20230206

摘要/Abstract

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400 μg，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 SDS-PAGE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-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 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。

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

Abstract:

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 isopropyl β-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 400 μg 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 Actrophozoite 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 SDS-PAGE 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∶6 400，and the concentrations of IgG1 and IgG2a were 116.76 g·L^－1 and 1 136.15 mg·L^－1， respectively.The Western blotting 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 with control group， the adhesion rate of Ac trophozoites to the Vero cells in the experiment group was significantly decreased（P<0.01） 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.

Key words: Acanthamoeba castellanii, Trophozoite, Cell adhesion, Immunogenicity, Immunoreactivity

中图分类号:

R382.1

李晶,杨舒越,赵佳欣,孔繁利,郭思瑶,冯宪敏. 卡氏棘阿米巴肌动蛋白1的免疫学特性和细胞黏附功能[J]. 吉林大学学报(医学版), 2023, 49(2): 308-314.

Jing LI,Shuyue YANG,Jiaxin ZHAO,Fanli KONG,Siyao GUO,Xianmin FENG. Immunological characteristics and cellular adhesion function of Acanthamoeba castellanii Actin 1[J]. Journal of Jilin University(Medicine Edition), 2023, 49(2): 308-314.

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