[摘 要] 目的：建立一种以乙型肝炎病毒核心蛋白（HBc）病毒样颗粒（VLP）为骨架，嵌合PD-1 B细胞表位的重组颗粒蛋白，研 究其对小鼠结直肠癌（CRC）的抑制效果。方法：设计PD-1 HBc VLP质粒并导入BL21（DE3）获得重组PD-1 HBc VLP表达菌株后， 通过优化培养温度，实现蛋白可溶性表达，经硫酸铵沉淀、Sepharose 4 Fast Flow尺寸排阻层析和DEAE阴离子树脂层析纯化获得高 纯度的PD-1 HBc VLP。采用电泳、WB、HPLC及电镜手段对其表达水平、蛋白纯度、颗粒形成率、形态特征及抗原结合活性等特征 进行系统表征，ELISA测定免疫小鼠后的血清效价，评价其免疫原性，构建CT26细胞CRC小鼠移植瘤模型，通过监测PD-1 HBc VLP 治疗后小鼠体质量和肿瘤体积变化，评估PD-1 HBc VLP抗肿瘤活性。结果：在28 ℃条件下，可溶性目的蛋白表达量占总蛋白的 10.27%，多步纯化后目的蛋白纯度为（91.77 ± 0.62）%，分子量为23 000，电镜测定粒径为（31.90 ± 1.88） nm，粒径仪测定粒径为（30.66 ± 0.62） nm，多分散指数（PDI）为（0.18 ± 0.05）。WB结果显示，PD-1 HBc VLP重组蛋白可分别与HBc VLP抗血清和PD-1抗体结合。 ELISA结果显示，经PD-1 HBc VLP 3次免疫后，第14天小鼠血清中总抗体效价为1∶640 000，其中抗PD-1的效价为1∶40 000。抗肿 瘤实验表明，PD-1 HBc VLP组小鼠的肿瘤抑制率为（27.99 ± 2.98）%，对照组在45 d全部死亡，PD-1 HBc VLP组中位生存期延长20 d。 结论：成功制备了以HBc VLP为骨架，嵌合PD-1 B细胞表位的PD-1 HBc VLP，且对小鼠结直肠癌有治疗效果。

[Abstract] Objective: To establish a recombinant particle protein using hepatitis B core protein (HBc) virus-like particles (VLPs) as the scaffold, with chimeric incorporation of PD-1 B cell epitopes, and to investigate its inhibitory effect on colorectal cancer in mice. Methods: A PD-1 HBc VLPs plasmid was designed and introduced into Escherichia coli BL21 (DE3) to obtain recombinant PD-1 HBc VLPs-expressing strains. Soluble protein expression was achieved by optimizing the culture temperature. Subsequently, high-purity PD-1 HBc VLPs were obtained via a series of purification steps, including ammonium sulfate precipitation, size-exclusion chromatography, and anion-exchange chromatography. The expression level, protein purity, particle formation efficiency, morphological characteristics, and antigen-binding activity of the obtained PD-1 HBc VLPs were systematically characterized by electrophoresis, WB, highperformance liquid chromatography (HPLC), and transmission electron microscopy. Immunogenicity was evaluated by ELISA to determine serum antibody titers in immunized mice. CT26 colorectal carcinoma cell transplant tumor model was constructed in mice, and the anti-tumor activity of PD-1 HBc VLPs was further assessed in vivo by comparing tumor growth, body weight and tumor volume changes. Results: At 28 ℃, the soluble target protein accounted for 10.27% of total protein. The target protein was purified using Sepharose 4 Fast Flow molecular sieve and DEAE anion resin chromatography, with a purity of (91.77 ± 0.62)% and a molecular weight of 23 000. The particle size measured by electron microscopy was (31.90 ± 1.88) nm, while particle size analyzer showed a particle size of (30.66 ± 0.62) nm, and the polydispersity index (PDI) was (0.18 ± 0.05). WB analysis demonstrated that the recombinant PD-1 HBc VLPs could bind to anti-HBc VLPs serum and anti-PD-1 antibodies, respectively. ELISA results showed that on day 14 after the third immunization with PD-1 HBc VLPs, the total antibody titer in mouse serum reached 1∶640 000, with an anti-PD-1 antibody titer of 1∶40 000. Anti-tumor experiments showed that PD-1 HBc VLPs achieved a tumor inhibition rate of (27.99 ± 2.98)% in mice with colorectal carcinoma. All mice in the control group died by day 45, whereas the median survival time (MST) in the PD-1 HBc VLPs group was prolonged by 20 days. Conclusion: PD-1 HBc VLPs containing PD-1 B cell epitopes were successfully prepared and demonstrated therapeutic efficacy against colorectal cancer in mice.

[基金项目] 2023年度第二批省级科技研发计划（235200810052）；2024年河南省科技攻关项目（242102311221）；河南省高 等学校重点科研项目（24B320020）