基于O型口蹄疫病毒VP2_(1-33)肽段增强VP1_(141-160)肽段免疫原性的新型纳米颗粒疫苗研究  

Increasing immune response of calcium phosphate nanoparticle prepared with VP1_(141-160) and VP2_(1-33) peptide of foot and mouth disease virus serotype O

作　　者：付丽新[1,2] 唐冬梅[1] 周彪[1] 刘香易[1] 罗又才 严亨秀 FU Li-xin;TANG Dong-mei;ZHOU Biao;LIU Xiang-yi;LUO You-cai;YAN Heng-xiu(College of Life Science and Technology,Southwest Minzu University,Chengdu 610017,China;Department of Dermatovenereology,Chengdu Second People＇s Hospital,Chengdu 610017,China)

机构地区：[1]西南民族大学生命科学与技术学院,成都610000 [2]成都市第二人民医院皮肤科,成都610017

出　　处：《中国人兽共患病学报》2018年第8期689-696,共8页Chinese Journal of Zoonoses

基　　金：四川省科技厅应用基础研究项目(No.18YYJC0228);中央高校基本科研业务费专项基金项目(No.2018NZD16);西南民族大学校级教改重大培育项目(No.2017ZDPY01)联合资助~~

摘　　要：目的研究O型口蹄疫病毒中VP2_(1-33)肽段能否增强VP1_(141-160)肽段的免疫原性及其可能的免疫机制。方法分别构建两种新的含有VP1和VP2的重组质粒(PcDNA3.1-VP1、PcDNA3.1-VP2),并将重组质粒制备成纳米颗粒,检测其体外转染效率。然后将上述纳米颗粒作为基因疫苗免疫Balb/c小鼠,PBS及空载质粒作为对照。液相阻断ELISA方法检测不同时间小鼠血清中抗体情况;流式细胞术和免疫组化染色的方法分析免疫小鼠外周血、脾脏和腹股沟淋巴结中CD4+、CD8+T淋巴细胞的差异;淋巴细胞增殖实验检测免疫28d后小鼠外周血中淋巴细胞的增殖能力。结果对重组质粒进行鉴定,结果显示重组质粒(PcDNA3.1-VP1、PcDNA3.1-VP2)构建成功;体外转染实验结果显示该纳米颗粒体外转染效率约为28%,具有较高的转染效率。ELISA检测显示VP1与VP2共同免疫小鼠血清中的抗体滴度均在初次免疫后的第21和28d达到最高,并显著高于对照组(F=4.625,P<0.05);流式和免疫组化染色结果分析发现VP1与VP2共同免疫小鼠血液、淋巴结和脾脏中CD4^+、CD8^+T淋巴细胞数均显著高于对照组;淋巴细胞增殖实验结果显示VP1与VP2共同免疫小鼠淋巴细胞增殖能力均显著高于对照组(F=12.73,P<0.05)。结论 O型FMDV VP2的1-33肽段能增强VP1的141-160肽段的免疫原性,其可能通过激活细胞免疫和体液免疫实现。We investigated whether the VP2 peptide could increase the immune responses by VP1 peptide and the possible immune-mechanism. Two confirmed constructs were named PcDNA3.1-VP1 and PcDNA3.1-VP2 separately. The plasmids DNA coated nanoparticles of calcium phosphate were prepared. The transfection of the HEK293 cells with the DNA entrapped into calcium phosphate nanoparticles or in free form was carried out with the fluorophor protein coding plasmid DNA CaPi-PcDNA3.1-GFP. The efficiency of transfection of 293HEK cells by the nanoparticle entrapped DNA was markedly higher than the control group. BALB/c mice were divided into four groups randomly. Group A was vaccinated with 200 μL sterility PBS as control. Group B was vaccinated with 20 μg CaPi-PcDNA3.1 vector as control. Group C was vaccinated with 20 μg CaPi-PcDNA3.1-VP1. Group D was vaccinated with 20 μg CaPi-PcDNA3.1-VP1＋20 μg CaPi-PcDNA3.1-VP2. The serum antibody of mice after immunized was measured by LPB-ELISA. Antibody titers of VP1 and VP2 treated group maintained high levels on 28th dpi and significantly higher than other groups （F=4.625,P〈0.05）. The numbers of CD4＋ and CD8＋ T lymphocytes in VP1 and VP2 treated group were both higher than other groups. Lymphocyte proliferation assay showed that the stimulation index （SI） in VP1 and VP2 treated group was significantly higher than other groups （F=12.73, P〈0.05）. The VP2 peptide of FMDV “O” could enhance the immune responses induced by VP1 peptide. Mechanism study suggested that VP2 peptide could enhance the immune response with VP1 peptide by activating the cellar and humoral immune.

关 键 词：口蹄疫 钠米颗粒 VP1 VP2 基因疫苗 

分 类 号：R392[医药卫生—免疫学]