关键词: 细粒棘球绦虫, Polo样激酶2, 原核表达, 重组蛋白, 免疫保护

Abstract: Objective　To prepare the recombinant Echinococcus granulosus Polo⁃like kinase 2 (rEgPLK2) protein and evaluate its immunoprotective efficacy against cystic echinococcosis, so as to provide insights into research and development of novel vaccines against echinococcosis. Methods　The Polo⁃like kinase (PLK) protein sequences were retrieved from 12 species in the NCBI protein database, including E. granulosus and E. multilocularis. Multiple sequence alignment was performed using the Clustal Omega program, and structural visualization and homology analysis were conducted using the ESPript 3.2 program. The recombinant plasmid pET⁃30a⁃EgPLK2 was transformed into BL21(DE3) competent cells. Protein expression was induced with isopropyl⁃β⁃D⁃thiogalactoside (IPTG), and sodium dodecyl sulfate⁃polyacrylamide gel electrophoresis (SDS⁃PAGE) was performed to characterize the expression and molecular weight of the rEgPLK2 protein. The purified rEgPLK2 protein was thoroughly emulsified with Freund's complete adjuvant at a 1 ∶ 1 volume ratio. Two New Zealand white rabbits were immunized with multi⁃point subcutaneous injection on the back at a dose of 300 μg per rabbit for primary immunization. For booster immunizations, the protein was emulsified with Freund's incomplete adjuvant at a 1 ∶ 1 volume ratio and administered on days 14, 28, and 42 after the primary immunization at a dose of 150 μg per rabbit. Serum was sampled from the rabbit ear vein on day 7 after the final immunization to yield anti⁃rEgPLK2 polyclonal antibodies. Antibody titer was determined by indirect enzyme⁃linked immunosorbent assay (ELISA), and antibody specificity was verified by Western blotting. The tissue localization of the EgPLK2 protein was detected in E. granulosus protoscoleces and adult worms using immunofluorescence assay (IFA). Eighteen 6⁃ to 8⁃week⁃old female SPF⁃grade BALB/c mice were randomly divided into three groups, including the blank control group, rEgPLK2⁃ISA immunization group, and PBS⁃ISA adjuvant control group, of 6 mice each group. Mice in the rEgPLK2⁃ISA immunization group and PBS⁃ISA group received three primary immunizations via intramuscular injection, and animals in the rEgPLK2⁃ISA immunization group was inoculated with immunogens prepared by emulsifying rEgPLK2 protein with ISA 201 adjuvant at a 1 ∶ 1 volume ratio (6 μg per mouse), while mice in the PBS⁃ISA adjuvant control group received an equal volume of PBS emulsified with ISA adjuvant at a 1 ∶ 1 volume ratio. A fourth booster immunization was administered via intraperitoneal injection. Mice in the rEgPLK2⁃ISA immunization group received a booster immunization with 8 μg of rEgPLK2 protein per mouse, and animals in the PBS⁃ISA group received an equal volume of PBS, with immunizations given at 2⁃week intervals. Mice in the blank control group were given no treatment, and housed under standard conditions. Tail vein blood was collected from all mice 7 days after the final immunization, and levels of specific anti⁃rEgPLK2 IgG antibody and its subclasses (IgG1, IgG2a, IgG2b, IgG3) were measured by indirect ELISA. E. granulosus infection was modelled in mice through injection with 1 000 E. granulosus protoscoleces via intrahepatic portal vein in the rEgPLK2⁃ISA immunization group and PBS⁃ISA adjuvant control group 2 weeks after the last immunization. All mice were sacrificed and dissected. The number of cysts was counted in mouse livers, and the cyst reduction rate was calculated. Liver tissues were processed for paraffin sectioning and stained with hematoxylin and eosin (HE), and histopathological changes were examined under a light microscope. Results　Sequence analysis revealed that EgPLK2 shared a high amino acid sequence homology with E. multilocularis PLK2 (EmPLK2) and contained the typical domains of the Polo⁃like kinase family, including the serine/threonine protein kinase catalytic domain (STKc) and Polo⁃box. The IPTG⁃induced rEgPLK2 protein was mainly expressed in the form of inclusion bodies, and the purified rEgPLK2 protein showed a relative molecular mass of approximately 70 kDa. The prepared rabbit anti⁃rEgPLK2 polyclonal antibody had a titer of 1 ∶ 256 000, and Western blotting assay showed that this antibody specifically recognized the rEgPLK2 protein with a relative molecular mass of approximately 70 kDa. Immunofluorescence assay showed that the EgPLK2 protein was localized in the excretory bladder and rostellum of E. granulosus protoscoleces, as well as the tegument, suckers, and inter⁃proglottid junctions of adult worms. Immunoprotective assay showed that the serum levels of specific anti⁃rEgPLK2 IgG, IgG1, IgG2a, and IgG2b antibodies were 2.92 ± 0.49, 0.33 ± 0.10, 0.31 (0.36), and 3.12 (1.73) in mice in the rEgPLK2⁃ISA immunization group, which were all significantly higher than those in the PBS⁃ISA adjuvant control group (0.14 ± 0.04, 0.07 ± 0.01, 0.12 ± 0.04, and 0.11 ± 0.04, respectively) (t = 19.28 and 8.46, Z = 3.75 and 4.15; all P values < 0.001); however, there was no significant difference in the serum anti⁃IgG3 antibody level between the rEgPLK2⁃ISA immunization group and the PBS⁃ISA adjuvant control group [0.07 (0.01) vs. 0.073 (0.07); Z = 0.69, P > 0.05)]. In the mouse model of E. granulosus infections, the area of hepatic lesions was reduced and the inflammatory infiltration was alleviated in the rEgPLK2⁃ISA immunization group than in the PBS⁃ISA adjuvant control group, and the number of hepatic cysts was higher in the PBS⁃ISA adjuvant control group than in the rEgPLK2⁃ISA immunization group [8.00 (2.00) vs. 1.00 (0.75); Z = -2.93, P < 0.01], with a cyst reduction rate of 80.40%. Indirect ELISA assay measured higher serum levels of specific anti⁃rEgPLK2 IgG (3.28 ± 0.48 vs. 0.11 ± 0.04; t = 15.86, P < 0.01), IgG1 (0.29 ± 0.02 vs. 0.09 ± 0.01; t = 15.67, P < 0.01), IgG2a [3.71 (1.09) vs. 0.08 (0.03); Z = 2.88, P < 0.01], and IgG2b antibodies [3.34 (1.01) vs. 0.08 (0.03); Z = 2.88, P < 0.01] in the rEgPLK2⁃ISA immunization group than in the PBS⁃ISA adjuvant control group, and there was no significant difference in the serum level of the specific anti⁃rEgPLK2 IgG3 antibody between the rEgPLK2⁃ISA immunization group and the PBS⁃ISA adjuvant control group (0.07 ± 0.01 vs. 0.07 ± 0.01; t = 1.29, P > 0.05). Conclusions　The prokaryotic expression system has been successfully constructed for the EgPLK2 gene and the anti⁃rEgPLK2 polyclonal antibody has been obtained. The rEgPLK2 protein exhibits a high immunogenicity, and is effective to protect against E. granulosus infection, and inhibits cyst development, which is a promising candidate vaccine target against cystic echinococcosis.

Key words: Echinococcus granulosus, Polo?like kinase 2, Prokaryotic expression, Recombinant protein, Immunoprotection