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

Abstract

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

CLC Number:

R382.1

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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Immunological characteristics and cellular adhesion function of Acanthamoeba castellanii Actin 1

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