特异性ETEC K99-SIgA免疫复合物增强小鼠黏膜免疫反应的研究  

Enhancement of intestinal mucosal immune response by ETEC K99-SIgA complex

作　　者：陈小燕 白永飞 张冰冰[1] 肖翠红[1] 王桂华[1] 刘卫贞 窦速林 侯喜林[2] 余丽芸[1] CHEN Xiao-yan;BAI Yong-fei;ZHANG Bing-bing;XIAO Cui-hong;WANG Gui-hua;LIU Wei-zhen;DOU Su-lin;HOU Xi-lin;YU Li-yun(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;College of Animal Science and Technology,Heilongjiang Baiyi Agricultural University,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国预防兽医学报》2021年第7期739-745,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：省自然科学基金面上项目(C2017047);科技厅自然科学基金委员会资助。

摘　　要：为探究产肠毒素性大肠杆菌(ETEC)特异性K99-SIgA免疫复合物对激发小鼠黏膜免疫反应的影响,本实验构建pET-32a-K99/BL21(DE3)原核表达载体,经IPTG诱导表达后以亲和层析方法纯化制备了重组K99蛋白(rK99);采用rK99蛋白和ETEC标准菌株K99经腹腔注射免疫BALB/c小鼠制备鼠源K99多克隆抗体;采用PBS溶解的ETEC K99菌液口服感染BALB/c小鼠,获得含有特异性SIgA抗体的粪便上清,以His pull-down制备得到特异性K99-SIgA复合物;利用特异性K99-SIgA复合物刺激ETEC K99口服感染小鼠后分离的脾淋巴细胞,分别采用ELISPOT检测细胞因子、CCK-8法检测小鼠脾T淋巴细胞增殖以及小鼠T淋巴细胞CTLL-2活力。结果显示:rK99在E. coli BL21(DE3)中高效表达,纯化蛋白浓度约4.5 mg/mL;腹腔免疫后的小鼠血清检出较高效价的K99多克隆抗体,腹腔免疫后的小鼠血清检出较高的K99多抗效价;以ETEC K99菌液口服感染小鼠获得大量的特异性SIgA抗体,抗体水平在感染后42 d达到高峰;制备获得特异性K99-SIgA复合物,浓度约为2 mg/mL,K99多抗能够有效地与特异性K99-SIgA复合物反应;特异性K99-SIgA复合物刺激小鼠脾淋巴细胞相比K99单独抗原能更有效地上调TNF-α、IFN-γ、IL-2、IL-4、IL-6、IL-5细胞因子分泌水平,促进小鼠脾T淋巴细胞及小鼠效应T细胞CTLL-2的增殖活化,表明特异性K99-SIgA免疫复合物能够有效地增强小鼠的黏膜免疫应答。本研究为免疫复合物口服疫苗的研制提供了参考依据。In order to explore the effect of K99-SIgA-specific immune complexes on stimulating mucosal immune response,the prokaryotic expression vector p ET-32 a-K99 was constructed, and the recombinant protein K99(r K99) was purified by affinity chromatography. BALB/c mice were immunized with r K99 and reference strain ETEC K99 by intraperitoneal injection to prepare the mouse K99 polyclonal antibodies. The BALB/c mice were orally infected with ETEC K99 solution dissolved in PBS, and the fecal supernatant containing specific SIg A antibodies was obtained. The K99-SIg A-specific complexes were prepared by His-Tag pull-down assay. The K99-SIg A-specific complexes were used to stimulate the spleen lymphocytes from the mice orally infected the ETEC K99. The cytokines were detected by ELISPOT, and the proliferation of splenic T lymphocytes and the activity of CTLL-2 of effector T cells were detected by CCK-8 method. The results showed that the recombinant protein of r K99 was highly expressed in E. coli BL21(DE3), and the concentration of the purified protein was about 4.5 mg/m L. The titers of polyclonal antibodies against K99 was detected in the serum of intraperitoneally immunized mice. A large amount of SIg A specific antibodies were obtained in mice infected orally with ETEC K99 bacterial solution, and the level of antibodies were reached their peak on days 42 post infection. The concentration of K99-SIg A complexes was about 2 mg/m L. The anti K99 polyclonal antibody could effectively react with K99-SIg A-specific complex. The secretiones of TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-5 were increased much more in the stimulation group of the K99-SIg A-specific complex than that in the K99 antigen group. The proliferation and activation of mouse spleen T lymphocytes and mouse effector T cells CTLL-2 could also be promoted by the K99-SIg A-specific complexes. It indicated that the K99-SIg A-specific immune complexes could effectively enhance the mucosal immune response in mice. This study provided a reference for the development

关 键 词：产肠毒素性大肠杆菌黏附性菌毛K99 SIGA 免疫复合物 免疫反应 

分 类 号：S852.4[农业科学—基础兽医学]