自组装纳米颗粒肿瘤疫苗OVA257-264-mi3的构建及其保护效果评价  

Construction of self-assembled nanoparticle tumor vaccine OVA 257-264-mi3 and evaluation of its protective efficacy

作　　者：陈源 高晨 李宇航 崔致远 程新 张怡[2] 于博 顾江[2] 杨宪[1] CHEN Yuan;GAO Chen;LI Yuhang;CUI Zhiyuan;CHENG Xin;ZHANG Yi;YU Bo;GU Jiang;YANG Xian(School of Life Sciences,Chongqing Normal University,Chongqing,401331;Department of Microbiology and Biochemical Pharmacy,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Orthopaedics,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]重庆师范大学生命科学学院,重庆401331 [2]陆军军医大学(第三军医大学)药学与检验医学系,重庆400038 [3]陆军军医大学(第三军医大学)第一附属医院骨科,重庆400038

出　　处：《陆军军医大学学报》2024年第12期1361-1368,共8页Journal of Army Medical University

基　　金：国家重点研发计划(2021YFC2301405)。

摘　　要：目的构建SpyCatcher-mi3纳米颗粒疫苗递送载体,评价其在增强卵清蛋白CD8+T细胞表位肽OVA 257-264免疫原性和在小鼠荷瘤模型中免疫保护效果。方法SpyCatcher-mi3蛋白由大肠杆菌表达,依次经亲和层析、阴离子交换层析纯化得到;合成OVA 257-264-SpyTag多肽,通过SpyTag/SpyCatcher蛋白质连接系统构建纳米颗粒OVA 257-264-mi3;通过细胞溶血实验和细胞毒性实验评价OVA 257-264-mi3体外安全性,记录小鼠体重变化和镜检脏器组织切片HE染色评价OVA 257-264-mi3体内安全性;将6~8周龄,18~20 g,SPF级,雌性C57BL/6小鼠按随机数字表法分为OVA 257-264-mi3组、OVA 257-264组和Control组,每组14只,分别于第0、14、28天免疫小鼠,末次免疫后第14天通过ELISpot检测其脾脏淋巴细胞中IFN-γ分泌细胞数量评价其免疫原性;在小鼠荷瘤模型上通过核磁共振成像等手段评价纳米颗粒疫苗OVA 257-264-mi3的保护效果。结果SpyCatcher-mi3和OVA 257-264-mi3均自组装形成均一稳定的纳米颗粒,平均粒径约为43.8 nm和91.3 nm;OVA 257-264-mi3在体内外均具有良好的安全性;OVA 257-264-mi3组小鼠每1×106个脾脏淋巴细胞中IFN-γ分泌细胞数量达253,显著高于OVA 257-264组(P<0.05);OVA 257-264-mi3组小鼠第22天荷瘤体积约151.1 mm 3,显著小于OVA 257-264组(P<0.05),且观察期内生存率达60%,显著高于OVA 257-264组(P<0.05)。结论成功构建了纳米颗粒疫苗OVA 257-264-mi3,其增强肿瘤抗原表位肽OVA 257-264免疫原性,在小鼠荷瘤模型中保护效果良好,为肿瘤新抗原疫苗的研究提供了理论基础。Objective To construct SpyCatcher-mi3 nanoparticle vaccine delivery vectors,evaluate their role in enhancing the immunogenicity of the ovalbumin CD8+T-cell epitope peptide,OVA 257-264,and determine its protective effect in a model which mice were immunized and subcutaneously challenged with E.G7-OVA tumor cells.Methods SpyCatcher-mi3 proteins were expressed by E.coli and purified by affinity chromatography and anion exchange chromatography sequentially.OVA 257-264-SpyTag peptide was obtained by synthesis.The OVA 257-264-mi3 nanoparticles were produced by the SpyTag/SpyCatcher system.The toxicity of OVA 257-264-mi3 was evaluated using hemolysis assay,CCK-8 assay and mouse experiment.A total of 42 female SPF-grade C57BL/6 mice(6~8 weeks old,18~20 g)were randomly divided into OVA 257-264-mi3,OVA 257-264,and control groups,with 14 mice in each group.Then the mice in each group were immunized on days 0,14 and 28.In 14 d after the last immunization,the amounts of spot-forming cells(SFCs,indicating IFN-γsecreting cells in splenic lymphocytes)were determined using ELISpot assay to evaluate their immunogenicity.After the immunized mice were subcutaneously implanted with E.G7-OVA tumor cells,the antitumor effect of the vaccine in prophylactic xenograft tumor model was evaluate by observing tumor volumes with a caliper and tumor growth with MRI.Results Both SpyCatcher-mi3 and OVA 257-264-mi3 could be self-assembled to form homogeneous and stable nanoparticles,with an average particle size of about 43.8 and 91.3 nm,respectively.The OVA 257-264-mi3 was safe for in vitro and in vivo toxicity evaluation.The number of IFN-γsecreting cells per 1×106 splenic lymphocytes reached 253 in the OVA 257-264-mi3 group of mice,significantly higher than that in the OVA 257-264 group and the Control group(P<0.05).The tumor volume of mice in the OVA 257-264-mi3 group was about 151.1 mm 3 on day 22,which was significantly smaller than that of the OVA 257-264 group and the Control group(P<0.05),and the survival rate during the observation pe

关 键 词：SpyCatcher-mi3 OVA 257-264 纳米颗粒疫苗 保护效果评价 

分 类 号：R392-33[医药卫生—免疫学] R392.7[医药卫生—基础医学] R979.19