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

摘要： 目的　制备细粒棘球绦虫（Echinococcus granulosus）Polo样激酶2（Polo⁃like kinase 2，PLK2）重组蛋白（recombinant E. granulosus PLK2，rEgPLK2），并评估其对细粒棘球蚴病的免疫保护效果，为棘球蚴病新型疫苗研发提供参考。方法　从NCBI蛋白质数据库获取细粒棘球绦虫和多房棘球绦虫等12个物种的Polo样激酶蛋白序列，采用在线工具Clustal Omega进行多重序列比对，通过ESPript 3.2服务器进行结构可视化及同源性分析。将pET⁃30a⁃EgPLK2重组表达质粒转化至BL21（DE3）感受态细胞中，经异丙基⁃β⁃D⁃硫代半乳糖苷（isopropyl⁃β⁃D⁃thiogalactoside，IPTG）诱导表达、电泳分析，鉴定rEgPLK2重组蛋白的表达形式及分子量。将纯化的rEgPLK2蛋白与弗氏完全佐剂按1 ∶ 1体积比充分乳化，采用背部多点皮下注射方式对2只新西兰大白兔进行首次免疫，剂量为300 μg/只；将rEgPLK2蛋白与弗氏不完全佐剂按1 ∶ 1体积比充分乳化，于首次免疫后第14、28、42天以背部多点皮下注射方式分别进行3次加强免疫，剂量为150 μg/只；末次免疫后第7天经兔耳静脉采血分离血清，得到抗rEgPLK2多克隆抗体。采用间接酶联免疫吸附（enzyme⁃linked immunosorbent assay，ELISA）法检测抗体效价，通过免疫印迹（Western blotting，WB）法鉴定抗体特异性。利用免疫荧光技术，观察EgPLK2蛋白在细粒棘球绦虫原头蚴和成虫中的组织定位。将18只6 ~ 8周龄雌性SPF级BALB/c小鼠随机分为空白对照组、rEgPLK2⁃ISA免疫组和PBS⁃ISA佐剂组，每组6只。rEgPLK2⁃ISA免疫组和PBS⁃ISA佐剂组小鼠均以肌肉注射方式进行3次基础免疫和1次加强免疫，每次免疫间隔2周。在基础免疫中，rEgPLK2⁃ISA免疫组接种rEgPLK2蛋白与ISA佐剂按1 ∶ 1体积比混合乳化的免疫原，剂量为6 μg/只；PBS⁃ISA佐剂组接种等体积PBS与ISA佐剂按1 ∶ 1体积比混合乳化的免疫原。在加强免疫中，rEgPLK2⁃ISA免疫组经腹腔注射rEgPLK2蛋白，剂量为8 μg/只；PBS⁃ISA佐剂组经腹腔注射等体积PBS。空白对照组不作处理。末次免疫后第7天，采集各组小鼠尾静脉血清，采用间接ELISA法测定小鼠血清中抗rEgPLK2特异性IgG抗体及其亚类IgG1、IgG2a、IgG2b、IgG3抗体水平。于末次免疫2周后，rEgPLK2⁃ISA免疫组和PBS⁃ISA佐剂组小鼠经肝门静脉注射1 000个原头蚴建立细粒棘球蚴感染模型，3个月后剖检小鼠，对各组小鼠肝脏包囊数量进行统计分析，并计算减囊率。对肝脏组织石蜡切片进行苏木素⁃伊红（hematoxylin and eosin，HE）染色，光学显微镜下观察组织病理学变化。结果　序列分析结果显示，细粒棘球绦虫与多房棘球绦虫PLK2蛋白氨基酸序列高度相似，含有Polo样激酶家族典型的结构域，包括丝氨酸/苏氨酸蛋白激酶催化结构域（STKc）和Polo⁃box。经IPTG诱导的rEgPLK2蛋白主要以包涵体形式存在，纯化后的rEgPLK2蛋白相对分子质量约为70 kDa。制备的兔抗rEgPLK2多克隆抗体效价达1 ∶ 256 000，WB检测结果显示，其可特异性识别相对分子质量约为70 kDa的rEgPLK2蛋白。免疫荧光实验结果显示，EgPLK2蛋白定位于细粒棘球绦虫原头蚴的后膀胱、顶突及成虫的表皮、吸盘和节片连接处。免疫保护实验结果显示，rEgPLK2⁃ISA免疫组小鼠血清抗rEgPLK2特异性IgG、IgG1、IgG2a、IgG2b抗体水平分别为2.92 ± 0.49、0.33 ± 0.10、0.31（0.36）、3.12（1.73），均高于PBS⁃ISA佐剂组（0.14 ± 0.04、0.07 ± 0.01、0.12 ± 0.04、0.11 ± 0.04），差异均有统计学意义（t = 19.28、8.46，Z = 3.75、4.15；P均< 0.001）；而rEgPLK2⁃ISA免疫组小鼠血清特异性IgG3［0.07（0.01）］与PBS⁃ISA佐剂组［0.07（0.07）］差异无统计学意义（Z = 0.69，P > 0.05）。在细粒棘球蚴感染模型中，rEgPLK2⁃ISA免疫组小鼠肝脏病灶面积较PBS⁃ISA佐剂组减小，炎症浸润减轻；PBS⁃ISA佐剂组小鼠肝脏包囊数量为8.00（2.00）个，rEgPLK2⁃ISA免疫组小鼠肝脏包囊数量减少至1.00（0.75）个（Z = −2.93，P < 0.01），减囊率为80.40%。间接ELISA结果显示，rEgPLK2⁃ISA免疫组小鼠血清抗rEgPLK2特异性IgG、IgG1、IgG2a、IgG2b抗体水平分别为3.28 ± 0.48、0.29 ± 0.02、3.71（1.09）、3.34（1.01），均高于PBS⁃ISA佐剂组［0.11 ± 0.04、0.09 ± 0.01、0.08（0.03）、0.08（0.03）］，差异均有统计学意义（t = 15.86、15.67，Z = 2.88、2.88；P均< 0.01）；而rEgPLK2⁃ISA免疫组小鼠血清特异性IgG3水平为0.07 ± 0.01，与PBS⁃ISA佐剂组（0.07 ± 0.01）差异无统计学意义（t = 1.29，P > 0.05）。结论　成功构建了EgPLK2基因的原核表达系统并获得了抗rEgPLK2多克隆抗体。rEgPLK2蛋白具有良好的免疫原性，可对抗细粒棘球蚴感染、遏制包囊发育，是一个具有潜力的抗细粒棘球蚴病疫苗候选靶点。

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