目的 建立嵌合表达恶性疟原虫环子孢子蛋白（PfCSP）的伯氏疟原虫（Pb）感染小鼠模型，并评估该模型在疫苗免疫保护效果评价中的应用价值，为推进针对CSP的疟疾疫苗研究提供可靠的概念验证平台。 方法 采用PCR鉴定和验证嵌合Pfcsp的转基因Pb虫株（PfCSP-Pb虫株），通过体外培养1~5 d、小鼠体内传代1~6 d比较PfCSP-Pb虫株与野生型Pb虫株在红内期的增殖、形态及原虫血症情况。以斯氏按蚊血餐分别感染PfCSP-Pb虫株或野生型Pb虫株3~4 d的ICR小鼠，按蚊感染后第14天，红汞染色观察按蚊中肠的卵囊情况，荧光显微镜检测中肠和唾液腺中子孢子的GFP荧光信号。收集感染PfCSP-Pb虫株或野生型Pb虫株的按蚊唾液腺子孢子，尾静脉注射感染ICR小鼠，感染42 h后，提取小鼠肝组织总RNA，qRT-PCR检测子孢子18S rRNA在小鼠肝期的相对表达量。用PfCSP特异性单克隆抗体mAB317（100 μg）被动免疫小鼠2 h后，用感染PfCSP-Pb虫株或野生型Pb虫株的按蚊唾液腺子孢子感染小鼠，qRT-PCR检测各组小鼠肝脏相对载虫量，验证mAB317单克隆抗体对PfCSP-Pb虫株子孢子感染肝细胞的阻断效果。 结果 PCR鉴定结果显示，PfCSP-Pb虫株稳定整合并表达pf-csp基因和gfp-luciferase报告基因。在红内期，PfCSP-Pb虫株体外和体内培养的红细胞原虫血症均与野生型Pb虫株的差异无统计学意义（F = 0.86、2.18，P > 0.05）；PfCSP-Pb虫株在环状体、滋养体与裂殖体阶段的形态与野生型Pb虫株一致，且胞质中可观察到稳定的GFP荧光蛋白表达。饲血后第14天，红汞染色显示，PfCSP-Pb虫株与野生型Pb均可在感染按蚊中肠形成大量卵囊，内含未成熟子孢子；蚊期感染第21天，在感染PfCSP-Pb虫株按蚊的唾液腺和中肠组织中检测到GFP荧光信号。肝期实验qRT-PCR结果显示，PfCSP-Pb虫株感染组中18S rRNA的相对表达量为1.26 ± 0.49，与野生型Pb虫株感染组的1.16 ± 0.67差异无统计学意义（t = 0.27，P > 0.05）。mAb317被动保护实验中，qRT-PCR检测结果显示，PfCSP‐Pb抗体组小鼠的肝脏相对载虫量为0.25 ± 0.48，低于PfCSP‐Pb对照组（未注射mAb317）小鼠的1.10 ± 0.51（t = 2.70，P < 0.05）；野生型Pb抗体组小鼠的肝脏相对载虫量为0.92 ± 0.44，与野生型Pb对照组的1.28 ± 0.69差异无统计学意义（t = 0.99，P > 0.05）。 结论 本研究建立了能稳定表达嵌合PfCSP的伯氏疟原虫感染小鼠模型。PfCSP-Pb虫株在体内外均保持稳定的生物学特性，并能特异性用于评估针对PfCSP抗体介导的免疫保护效果，为恶性疟疫苗开发的临床前研究提供了关键的技术平台和评价工具。

Objective To establish a mouse model of infection with Plasmodium berghei (Pb) chimerically expressing P. falciparum circumsporozoite protein (PfCSP), and to evaluate the value of the model in evaluation of vaccine-induced immunoprotective effectiveness, so as to provide a reliable proof-of-concept platform for advancing CSP-based malaria vaccines. Methods PCR assay was employed to characterize and verify the transgenic Pb strain chimerically expressing the Pfcsp gene (PfCSP-Pb strain). The proliferation, morphology, and parasitemia were compared between the PfCSP-Pb and wild-type Pb strains during the erythrocytic stage through in vitro culture for 1 to 5 days and in vivo passage in mice for 1 to 6 days. ICR mice that had been infected with either the PfCSP-Pb strain or the wild-type Pb strain for 3 to 4 days were fed to Anopheles stephensi mosquitoes. The oocysts were observed in mosquito midguts using mercurochrome staining 14 days post-infection with An. stephensi, and green fluorescent protein (GFP) fluorescence signals of sporozoites were detected in mosquito midguts and salivary glands using fluorescence microscopy. Salivary gland sporozoites from Anopheles mosquitoes infected with either the PfCSP-Pb strain or the wild-type Pb strain were collected and intravenously injected into ICR mice via tail vein. Mice were sacrificed 42 hours post-infection, and mouse liver tissues were sampled. Total RNA was extracted from mouse liver tissues, and the relative expression of sporozoite 18S rRNA was detected in mice during the liver stage using real-time quantitative real-time reverse transcription PCR (qRT-PCR) assay. Following passive immunization with the PfCSP-specific mAB317 monoclonal antibody (100 μg) in mice, mice were infected with salivary gland sporozoites from Anopheles mosquitoes infected with either the PfCSP-Pb strain or the wild-type Pb strain. Mouse liver parasite load was detected using qRT-PCR assay to verify the blocking effect of the mAB317 monoclonal antibody on the infection of hepatocytes with sporozoites of the PfCSP-Pb strain. Results PCR assay confirmed the stable integration and expression of the pf-csp gene and the gfp-luciferase reporter gene by the PfCSP-Pb strain. No significant differences were observed in proliferation rates between the PfCSP-Pb strain and the wild-type Pb strain either in in vitro (F = 0.86, P > 0.05) or in vivo (F = 2.18, P > 0.05) during the erythrocytic stage. The PfCSP-Pb strain exhibited identical morphology to the wild-type strain across the ring, trophozoite, and schizont stages, with stable GFP fluorescence protein expression observed in the cytoplasm. Mercurochrome staining revealed that both the PfCSP-Pb strain and the wild-type Pb strain produced a large number of oocysts in the midguts of Anopheles mosquitoes infected with both the PfCSP-Pb strain and the wild-type Pb strain on day 14 post-infection, which contained sporozoite precursor cells, and GFP fluorescence signals were detected in the salivary glands and midgut tissues of Anopheles mosquitoes infected with the PfCSP-Pb strain. In the liver stage, qRT-PCR assay showed that the relative 18S rRNA expression was 1.26 ± 0.49 in the PfCSP-Pb strain infection group, which was comparable to that in the wild-type Pb infection group (1.16 ± 0.67) (t = 0.27, P > 0.05). Following immunization of the mAB317 monoclonal antibody, the relative liver parasite load was lower in the PfCSP-specific monoclonal antibody treatment group (0.25 ± 0.48) than in the control group (1.10 ± 0.51) (t = 2.70, P < 0.05) in mice infected with salivary gland sporozoites from Anopheles mosquitoes infected with the PfCSP-Pb strain, while no significant difference was seen in the relative liver parasite load between the PfCSP-specific monoclonal antibody treatment group (0.92 ± 0.44) and the control group (1.28 ± 0.69) (t = 0.99, P > 0.05) in mice infected with salivary gland sporozoites from Anopheles mosquitoes infected with the wild-type Pb strain. Conclusion This study successfully established a chimeric Pb infection model stably expressing chimeric PfCSP. The PfCSP-Pb strain maintains stable biological characteristics both in vitro and in vivo, and is specifically able to be used to evaluate antibody-mediated immunoprotection against PfCSP, which provides a key technical platform and evaluation tool for preclinical researches on development of P. falciparum malaria vaccines.