自制ISCOMATRIX与CpG联合的HCA587蛋白疫苗的制备及其免疫效应评估  

Preparation of HCA587 Protein Vaccine Combined with CpG and Self-Made ISCOMATRIX and Evaluation of its Immune Effect

作　　者：谭媛芳 陈惠媛 焦薇 范秋颖 邵国青[2] 熊祺琰[2] 陈娟娟[1] TAN Yuan-fang;CHEN Hui-yuan;JIAO Wei;FAN Qiu-ying;SHAO Guo-qing;XIONG Qi-yan;CHEN Juan-juan(Jiangxi Key Laboratory of Laboratory Medicine,Department of Clinical Laboratory,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Key Laboratory of Animal Diseases Diagnostic and Immunology,Ministry of Agriculture,National Center for Engineering Research of Veterinary Bio-products,Nanjing 210014,China)

机构地区：[1]南昌大学第二附属医院检验科,江西省检验医学重点实验室,南昌330006 [2]江苏省农业科学院兽医研究所,农业部动物疫病诊断与免疫重点开放实验室,国家兽用生物制品工程技术研究中心,南京210014

出　　处：《南昌大学学报（医学版）》2023年第5期1-7,F0003,共8页Journal of Nanchang University：Medical Sciences

基　　金：国家自然科学基金(82160314,81960497)。

摘　　要：目的确定自制免疫刺激复合物(ISCOMATRIX)作为HCA587蛋白疫苗佐剂的有效剂量,并探讨其与CpG联合作为HCA587蛋白疫苗佐剂的免疫效力和肿瘤治疗效果。方法应用透析法将Quil A、胆固醇和磷脂制备成ISCOMATRIX,经磷钨酸负染并采用透射电子显微镜观察自制ISCOMATRIX的结构。建立小鼠免疫模型,将联合CpG的HCA587蛋白疫苗与不同剂量的自制ISCOMATRIX佐剂混合,经尾根部皮下免疫小鼠;采用酶联免疫斑点试验(ELISpot)检测产生IFN-γ的脾细胞频数,采用酶联免疫吸附法(ELISA)检测血清HCA587特异性抗体水平。在小鼠右侧胁腹部皮下接种B16-HCA587肿瘤细胞建立荷瘤小鼠模型,按照处理方式不同将小鼠分为HCA587蛋白疫苗治疗组(接种100μL包含CpG与最佳剂量的自制ISCOMATRIX的HCA587蛋白疫苗)与PBS对照组(注射100μL的无菌PBS),使用游标卡尺测量肿瘤大小。结果自制ISCOMATRIX佐剂呈典型的蜂窝状结构,直径20~30 nm,大小均一。12、36μg(QuilA浓度分别为0.012%、0.036%)的自制ISCOMATRIX组小鼠脾细胞分泌IFN-γ的频数与商品化ISCOMATRIX阳性对照组比较,差异无统计学意义(均P>0.05),且显著高于CpG+36μg ISCOM对照组与PBS组(均P<0.0001);5μg与18μg自制ISCOMATRIX组小鼠分泌IFN-γ的脾细胞频数,高于CpG+36μg ISCOM对照组与PBS组(均P<0.0001),但低于商品化ISCOM组(P=0.004)。12、18与36μg自制ISCOMATRIX组的血清HCA587抗体水平与商品化ISCOM阳性对照组比较,差异无统计学意义(均P>0.05);与PBS组相比,12μg自制ISCOMATRIX组1×10^(4)倍稀释血清的抗体滴度显著升高(P=0.0062)。与PBS对照组相比,HCA587蛋白疫苗治疗组肿瘤生长速度减缓,且第35天测量时肿瘤大小显著较小(P=0.0181)。结论以12μg自制ISCOMATRIX作为佐剂联合CpG制备的HCA587蛋白疫苗能诱导有效的免疫应答,并抑制荷瘤小鼠的肿瘤的生长,具有一定的肿瘤治疗作用。Objective To determine the effective dose of self-made immunostimulatory complex(ISCOMATRIX)as an adjuvant for HCA587 vaccine,and to explore the immune efficacy and anti-tumor effect of its combination with CpG as adjuvants for HCA587 protein vaccine.Methods ISCOMATRIX was prepared from Quil A,cholesterol and phospholipids by dialysis,and its structure was observed by transmission electron microscope after negative staining with phosphotungstic acid.The immune model of mice was established,and the HCA587 protein combined with CpG was mixed with different doses of self-made ISCOMATRIX adjuvant to immunize C57BL/6J mice subcutaneously through the tail root.The splenocytes producing IFN-γwere detected by enzyme-linked immunosorbent spot assay(ELISpot),and the levels of serum anti-HCA587 specific antibody were measured by enzyme-linked immunosorbent assay(ELISA).The model of tumor-bearing mice was established by subcutaneous inoculation of B16-HCA587 tumor cells into the right flank of C57BL/6J mice.According to the different treatment methods,the mice were divided into treatment group(inoculated with 100μL HCA587 protein containing CpG and the optimal dose of self-made ISCOMATRIX)and control group(injected with 100μL sterile PBS solution).The tumor size was measured with vernier caliper.Results The self-prepared ISCOMATRIX adjuvant showed a typical honeycomb structure with a diameter of 20-30 nm and uniform size.The frequency of splenocytes secreting IFN-γin self-made 12 and 36μg ISCOMATRIX groups(QuilA concentrations were 0.012%and 0.036%,respectively)was not significantly different from that in commercial positive control group(P>0.05),but was significantly higher than that in adjuvant CpG+36μg ISCOM control group and PBS group(P<0.0001).The frequency of splenocytes secreting IFN-γin self-made 5 and 18μg ISCOMATRIX groups was higher than that in adjuvant CpG+36μg ISCOM group and PBS group(P<0.0001),but lower than that in commercial ISCOM group(P=0.004).The anti-HCA587 antibody levels in the self-made 12,1

关 键 词：免疫刺激复合物(ISCOMATRIX) 自行制备 CPG HCA587蛋白疫苗 免疫效应 

分 类 号：R392.9[医药卫生—免疫学]