目的 探讨刚地弓形虫棒状体蛋白18（ROP18）、中心蛋白家族蛋白TGME49_237490、卵囊壁特异性蛋白直系同源物TGME49_268230和微线体蛋白13（MIC13）四抗原融合蛋白（简称4 ×）的免疫保护作用。方法 PCR扩增获得4 × 目的基因，构建重组质粒并进行鉴定。将构建成功的质粒转化至大肠埃希菌BL21感受态细胞，进行目的蛋白的诱导表达和亲和纯化，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与蛋白免疫印迹分析（Western blotting）鉴定目的蛋白表达情况。36只雌性BALB/c小鼠随机分成免疫组与对照组，各18只，免疫组注射4 × 蛋白加弗氏佐剂，对照组注射等量PBS加弗氏佐剂，共免疫3次，每次间隔2周。分别于免疫后第0、2、4、6周采集小鼠眼眶血，分离血清，ELISA检测血清中IgG抗体水平，第6周加测IgG抗体亚型水平。末次免疫后2周，每组各取10只小鼠，无菌取脾，制备脾细胞悬液，5只小鼠采用细胞增殖及毒性检测试剂盒检测细胞增殖水平，5只小鼠通过微球免疫法流式多因子检测试剂盒检测细胞因子水平。末次免疫后2周，每组各取5只小鼠外周血，流式细胞术检测小鼠T细胞亚群比例。末次免疫后2 周，每组各取8只小鼠，腹腔注射100个弓形虫RH株速殖子攻击感染，3只小鼠通过荧光定量PCR检测脏器弓形虫负荷量，5只小鼠记录存活时间，绘制生存曲线，采用GraphPad Prism 8软件进行统计学分析。结果 4 × 基因PCR扩增片段约为3 576 bp，与预期大小一致。重组质粒经PCR、酶切鉴定均获得预期大小的条带。目的蛋白被成功表达、纯化，SDS-PAGE与Western blotting均在相对分子质量（M_r）110 000处可见明显条带。ELISA检测结果显示，免疫组IgG抗体水平在免疫后第0、2、4、6周的吸光度（A₄₅₀值）分别为0.079 ± 0.004、0.759 ± 0.179、1.670 ± 0.243、2.461 ± 0.056，对照组分别为0.080 ± 0.006、0.067 ± 0.009、0.080 ± 0.014、0.076 ± 0.011，免疫组除第0周（t = 0.05，P > 0.05）外均高于对照组（t = 9.88、23.33、34.66，均P < 0.05）；免疫后第6周，免疫组IgG1和IgG2a抗体亚型水平的A₄₅₀值分别为3.202 ± 0.401、3.725 ± 0.066，均高于对照组的0.082 ± 0.003、0.059 ± 0.017（t = 13.73、96.83，均P < 0.05）。流式细胞术检测结果显示，免疫组细胞因子γ干扰素（IFN-γ）、白细胞介素-2（IL-2）、IL-12、IL-4、IL-10水平分别为（4 998.52 ± 2 131.24）、（5.76 ± 1.02）、（1.38 ± 0.86）、（28.83 ± 1.64）、（3 376.57 ± 218.48）pg/ml，均高于对照组的（8.90 ± 0.17）、（3.05 ± 0.50）、（0.06 ± 0.13）、（11.18 ± 1.58）、（13.87 ± 3.55）pg/ml（t = 4.89、4.61、3.06、15.93、30.30，均P < 0.05）；免疫组小鼠的脾细胞增殖水平（3.286 ± 0.552）高于对照组（1.251 ± 0.157）（t = 9.15，P < 0.05）。免疫组CD3⁺CD4⁺T细胞和CD3⁺CD8⁺T细胞的比例分别为（31.15 ± 2.83）%、（18.06 ± 2.82）%，高于对照组的（26.82 ± 1.17）%、（9.33 ± 1.22）%（t = 3.16、6.36，均P < 0.05）。qPCR结果显示，免疫组小鼠的肝、脾、肺虫荷量分别为（206.7 ± 16.0）、（100.2 ± 15.4）、（7.1 ± 2.0）fg/mg，低于对照组的（20 086.0 ± 1 310.0）、（26 254.0 ± 9 658.0）、（5 300.0 ± 741.2）fg/mg（t = 26.29、4.69、12.37，均P < 0.05）。攻击感染后，对照组小鼠在8 d内均死亡，免疫组小鼠在30 d的观察期内，存活1只，存活时间显著延长（χ² = 7.47，P < 0.05）。结论 四抗原融合蛋白能在小鼠体内诱导产生有效的体液免疫和细胞免疫反应，具有显著的抗弓形虫感染保护效果。

Objective To investigate the immunoprotective effect of a four-antigen fusion protein vaccine (referred to as 4 ×) composed of Toxoplasma gondii rhoptry protein 18 (ROP18), centrin family protein TGME49_237490, oocyst wall-specific protein ortholog TGME49_268230, and microneme protein 13 (MIC13). Methods The 4 × target gene was amplified using PCR assay, and recombinant plasmids were constructed and characterized. The successfully constructed plasmids were transformed into Escherichia coli BL21 competent cells for induced expression and affinity purification of the target protein, and the expression of the target protein was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Thirty-six female BALB/c mice were randomly divided into an immunization group and a control group, of 18 mice in each group. Mice in the immunization group were injected with 4 × protein and Freund’s adjuvant, and animals in the control group were administered with phosphate-buffered saline and Freund’s adjuvant. Immunizations were administered three times at two-week intervals. Blood samples were collected from the orbital sinus 0, 2, 4, 6 weeks following immunization, and serum samples were isolated. The levels of serum IgG antibodies were measured using ELISA. In the final detection, the levels of IgG antibody subtypes were also assessed. Two weeks after the final immunization, ten mice from each group were sacrificed, and spleens were aseptically harvested to prepare splenocyte suspensions. Splenocyte suspensions from five mice were used to measure cell proliferation using the CCK8 assay, while those from the other five mice were used for analysis of cytokine levels using flow cytometry. Mouse peripheral blood was sampled two weeks following final immunization, and proportions of mouse T cell subsets were detected using flow cytometry in five mice from each group. Eight mice were sampled from each group two weeks following final immunization and challenged intraperitoneally with 100 tachyzoites of the T. gondii RH strain. Then, the burdens of T. gondii in organs of 3 mice were examined using real-time quantitative fluorescence PCR (qPCR) assay, and the survival of other 5 mice were monitored daily and survival curves were plotted, statistical analysis was performed using GraphPad Prism 8 software. Results The PCR amplification product of the 4 × gene was 3 576 bp, which was consistent with the expected size. Recombinant plasmids showed expected band sizes upon characterization with PCR assay and enzymatic digestion. The target protein was successfully expressed and purified, with prominent bands observed at M_r 110 000 by both SDS-PAGE and Western blotting. ELISA results showed that the IgG antibody levels (A₄₅₀ values) in the immunized group at weeks 0, 2, 4, and 6 post-immunization were 0.079 ± 0.004, 0.759 ± 0.179, 1.670 ± 0.243, and 2.461 ± 0.056, respectively. The corresponding values for the control group were 0.080 ± 0.006, 0.067 ± 0.009, 0.080 ± 0.014, and 0.076 ± 0.011, respectively. Except for week 0 (t = 0.05, P > 0.05), the IgG levels in the immunized group were significantly higher than those in the control group (t = 9.88, 23.33, 34.66; all P < 0.05). At week 6 post-immunization, the absorbance values of IgG1 and IgG2a antibodies were 3.202 ± 0.401 and 3.725 ± 0.066 in the immunization group and 0.082 ± 0.003 and 0.059 ± 0.017 in the control group (t = 13.73, 96.83; both P < 0.05), respectively. Flow cytometry detected that the levels of IFN-γ, IL-2, IL-12, IL-4, and IL-10 were (4 998.52 ± 2 131.24), (5.76 ± 1.02), (1.38 ± 0.86), (28.83 ± 1.64), and (3 376.57 ± 218.48) pg/ml in the immunization group and (8.90 ± 0.17), (3.05 ± 0.50), (0.06 ± 0.13), (11.18 ± 1.58), (13.87 ± 3.55) pg/ml in the control group (t = 4.89, 4.61, 3.06, 15.93, 30.30; all P < 0.05), respectively, and the proliferation level of splenocytes was 3.286 ± 0.552 in the immunization group and 1.251 ± 0.157 in the control group (t = 9.15, P < 0.05). The proportions of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were (31.15 ± 2.83)% and (18.06 ± 2.82)% in the immunization group and (26.82 ± 1.17)% and (9.33 ± 1.22)% in the control group (t = 3.16, 6.36; both P < 0.05), respectively. RT-qPCR assay detected that parasite burdens were (206.7 ± 16.0), (100.2 ± 15.4), and (7.1 ± 2.0) fg/mg in the mouse liver, spleen, and lungs in the immunization mice, and (20 086.0 ± 1 310.0), (26 254.0 ± 9 658.0) and (5 300.0 ± 741.2) fg/mg in the control group (t = 26.29, 4.69, 12.37; all P < 0.05). All mice in the control group died within 8 days following challenge, in the immunized group, only one mouse survived throughout the 30 d observation period, with a significantly prolonged survival time (χ² = 7.47, P < 0.05). Conclusion The four-antigen fusion protein vaccine may induce effective humoral and cellular immune responses in mice and present a significant protective effect against acute T. gondii infection.