牛环形泰勒虫烯醇化酶重组蛋白对小鼠免疫应答的研究  

Study on Immune Response of Recombinant Protein of Theileria annulata Enolase in Mice

作　　者：郑会珍 甘露 刘燕 何文文 葛晓敏 李永畅[1] 巴音查汗[1] 郭庆勇[1] ZHENG Hui-zhen;GAN Lu;LIU Yan;HE Wen-wen;GE Xiao-min;LI Yong-chang;BAYIN Cha-han;GUO Qing-yong(College of Veterinary Medicine,Xingjiang Agricultural University,Urumqi,Xingjiang,830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《动物医学进展》2024年第1期24-29,共6页Progress In Veterinary Medicine

基　　金：新疆维吾尔自治区区域协同创新专项项目(2021E01001)。

摘　　要：为了评估牛环形泰勒虫(Theileria annulata)烯醇化酶(enolase)原核重组蛋白对实验动物体液免疫水平和细胞免疫水平的影响,用His纯化柱和超滤管对pET-32a-enolase进行纯化和浓缩,应用Western blot分析蛋白的反应原性;另外利用牛环形泰勒虫烯醇化酶重组蛋白(r Ta-enolase)免疫Balb/c鼠制备多克隆抗体,经间接ELISA检测血清中重组蛋白的特异性抗体水平。免疫后,取小鼠脾脏制备淋巴细胞悬液,用流式细胞术检测脾脏T细胞亚群情况;用MTT试剂盒检测体外刺激后T淋巴细胞增殖情况;用Spss ANOVA单因素检验进行差异显著性分析。Western blot表明,enolase重组蛋白可与阳性血清发生特异性反应。ELISA检测免疫鼠特异性IgG/IgG1和IgG2a抗体均与对照组差异极显著(P<0.01);流式细胞术检测表明,免疫组(2.53±0.11)与对照组(1.85±0.11)CD4^(+)/CD8^(+)T细胞的比值差异极显著(P<0.01);用r Ta-enolase重组蛋白刺激小鼠脾脏淋巴细胞后明显促进其增殖(P<0.05)。研究结果表明r Ta-enolase免疫小鼠能够诱导小鼠产生极高的体液免疫水平以及细胞免疫应答水平,细胞免疫应答偏向于CD4^(+)T细胞,为后续其在疫苗中的免疫功能提供数据资料。The humoral immunity level and cellular immunity level of experimental animals were evaluated by using the prokaryotic recombinant protein of Theileria annulata enolase(2-phospho-D-glyceratehydrolyase,enolase).Then the reactivity of r Ta-enolase was also estimated by using Western blot after r Ta-enolase was purified and concentrated using“His”purification column and ultrafiltration tube.In addition,polyclonal antibodies of r Ta-enolase were prepared due to immunized Balb/c mice,and the specific antibody level of r Ta-enolase was measured by indirect ELISA.The splenic T-cell subpopulations were also tested by flow cytometry which were used for prepared lymphocyte suspensions of immunized mouse spleens.Furthermore,proliferation of lymphocytes was examined by MTT kit after in vitro stimulation.The data were statistically analyzed by one-way ANOVA.Western blot analysis proved r Ta-enolase can specifically react with positive sera,while the significant differences(P<0.01)were observed in ELISA.The ratio of CD4^(+)/CD8^(+)T cells in the immunized group(2.53±0.11)were significant differences(P<0.01)compared with control group(1.85±0.11).Proliferation profile of spleen lymphocytes were significant induced after mouse spleen lymphocytes was stimulated by r Ta-enolase(P<0.05).The results of this study indicated that high level of humoral immunity and cellular immune response was identified after r Ta-enolase immunization of mice,especially CD4^(+)T cell.This study will be helpful for its subsequent immune function in vaccines.

关 键 词：牛环形泰勒虫 烯醇化酶 特异性抗体水平 CD4^(+)T细胞 

分 类 号：S852.723[农业科学—基础兽医学] S852.42[农业科学—兽医学]