新疆出血热病毒糖蛋白多表位嵌合肽基因的免疫原性研究  

Immunogenicity of chimeric peptide gene of Xinjiang hemorrhagic fever virus glycoprotein

作　　者：美丽排提·玉素甫 王颖[1] 阿布力米提·莫明 李轶杰[1] 张渝疆 孙素荣[1] Meilipaiti·Yusufu;Wang Ying;Abulimiti·Moming;Li Yijie;Zhang Yujiang;Sun Surong(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China;Xinjiang Disease Prevention and Control Center,Urumqi 830002,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源与基因工程重点实验室,乌鲁木齐830046 [2]新疆维吾尔自治区疾病预防与控制中心,乌鲁木齐830002

出　　处：《国际生物医学工程杂志》2022年第1期15-23,共9页International Journal of Biomedical Engineering

基　　金：新疆高校科研计划自然科学重点项目(XJEDU2019I002);国家自然科学基金(81960369,81760365)。

摘　　要：目的探讨新疆出血热病毒(XHFV)糖蛋白的2个抗原优势区(GcⅠ和GcⅡ)以不同嵌合策略以及不同免疫方式诱导小鼠免疫应答的效果。方法以在5’’端加入或不加鼠白细胞介素-2(IL-2)信号肽与通用型辅助性T细胞(Th)表位为不同设计策略,将GcⅠ、GcⅡ及GcⅠ上已鉴定出来的表位(Gc^(233~248)、Gc^(241~256)和Gc^(281~296))分别进行融合并构建至真核表达载体pVAX1与原核表达载体pET-28a上。采用间接免疫荧光法检测重组真核质粒的体外表达效果后进行大量提取,将重组原核质粒转化大肠杆菌BL21进行诱导表达及纯化。通过基因免疫、蛋白免疫和DNA初免-蛋白加强免疫(联合免疫)3种方式免疫BALB/c小鼠,酶联免疫吸附测定法检测小鼠体内IgG抗体水平以及小鼠脾细胞培养上清中IL-4和干扰素-γ(IFN-γ)的含量,噻唑蓝比色法检测小鼠脾T淋巴细胞的增殖情况。结果双酶切及测序结果表明8种重组质粒构建成功。荧光显微镜下可见构建的重组真核质粒在体外细胞均能成功表达。3次免疫小鼠后,各实验组的小鼠血清IgG抗体水平和刺激指数均明显高于对照组(均P<0.01)。Th2型细胞因子IL-4与Th1型细胞因子IFN-γ的质量浓度检测结果表明,联合免疫组引起的免疫应答偏向Th1型。结论成功构建了XHFV糖蛋白的多表位嵌合疫苗且靶抗原在小鼠体内可有效表达,各免疫组与对照组相比均激发了较强的体液免疫及细胞免疫应答,其中pVAX1-ST(GcⅠe+GcⅡ)联合重组蛋白r(GcⅠe+GcⅡ)的免疫效果最好,有望作为XHFV的新型候选疫苗。Objective To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus(XHFV)glycoprotein.Methods The 5'end was added or not added with interleukin-2(IL-2)signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies.GcⅠand GcⅡand the epitopes previously identified on GcⅠ(Gc^(233-248),Gc^(241-256) and Gc^(281-296))were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a.The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified,and the recombinant eukaryotes were extracted by indirect immunofluorescent assay.BALB/c mice were immunized by protein immunity,gene immunity,and DNA prime-protein boost immunity.The IgG antibody level was measured by ELISA.The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen.Results The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed,and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy.After three times of immunization,the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group(P<0.01).The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma(IFN-γ)revealed that the response of the DNA prime-protein boost immunity was biased to Th1.Conclusions The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed,and the target antigen could be expressed effectively in vivo.The immune groups stimulated stronger humoral and cellular immune responses compared with the control group.Among them,the immune effect of pVAX1-ST(GcⅠe+GcⅡ)combined with recombinant protein r(GcⅠe+GcⅡ)was the best,and it is expected to be a new candidate vaccine for XH

关 键 词：新疆出血热病毒 糖蛋白 多表位疫苗 DNA初免-蛋白加强免疫策略 

分 类 号：R392[医药卫生—免疫学]