沙门菌侵袭蛋白SipA的抗体制备及其在感染小鼠组织和巨噬细胞内的表达特点  

Preparation of polyclonal antibody and expression characteristics of Salmonella invasion protein SipA in infected mouse tissues and macrophages

作　　者：何珍 田棋 肖红 黄鑫淼 廖卫东 姚敏[1] 黄廷华[1] HE Zhen;TIAN Qi;XIAO Hong;HUANG Xinmiao;LIAO Weidong;YAO Min;HUANG Tinghua(College of Animal Sciences,Yangtze University,Jingzhou 434025,China)

机构地区：[1]长江大学动物科学学院,湖北荆州434025

出　　处：《畜牧与兽医》2023年第8期64-72,共9页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31902231和31402055);长江大学教研项目(JY2020125)。

摘　　要：旨在研究沙门菌侵袭蛋白SipA的表达特点。利用PCR技术从沙门菌中扩增SipA基因,然后将该基因克隆到原核表达载体pET-28a,转入至大肠杆菌BL21菌株,经诱导剂IPTG诱导表达重组蛋白SipA,纯化后的融合蛋白SipA免疫大耳白兔获得SipA抗体,利用沙门菌14028感染小鼠以及小鼠巨噬细胞Raw264.7后,通过免疫组化和免疫荧光鉴定SipA表达部位以及SipA在巨噬细胞内的分布。结果:成功表达重组蛋白SipA,纯化的SipA抗体通过间接ELISA法和Western blot检测其效价可达到1∶512 000,并具有较强特异性。免疫组化结果显示SipA在感染小鼠的空肠、肠系膜淋巴结、脾脏、肝脏、肺以及结肠均有表达,尤其在淋巴细胞和巨噬细胞的组织部位表达显著。细胞免疫组化以及细胞免疫荧光结果进一步揭示毒力蛋白SipA在Raw 264.7细胞的细胞质有大量表达,并且转位至细胞核。The aim of this study was to investigate the expression characteristics of Salmonella 14028 virulence protein SipA by hybridization with polyclonal antibody SipA.The SipA gene was amplified from Salmonella by PCR,cloned into the prokaryotic expression vector PET-28A,and transferred into an Escherichia coli BL21 strain.Recombinant SipA protein was induced by IPTG,and the purified fusion protein SipA was immunized into white rabbits to obtain SipA antibodies.Then,mice and Raw264.7 cells were infected with Salmonella 14028 and the expression site of SipA and its distribution location in macrophages were identified by immunohistochemistry,cellular immunohistochemistry and cellular immunofluorescence.The results were as follows:The recombinant SipA protein was successfully expressed.The purified SipA antibody showed a titer of 1:512000 by indirect ELISA and Western blot,and it possessed strong specificity.The immunohistochemical results showed that SipA was expressed in the jejunum,mesenteric lymph nodes,spleen,liver,lung and colon of the infected mice,especially in their lymphocytes and macrophages.The results of immunohistochemistry and immunofluorescence showed that the virulence protein SipA was highly expressed in the cytoplasm of the Raw 264.7 cells and translocated to the nucleus.

关 键 词：沙门菌14028 毒力蛋白SipA 多克隆抗体 表达特征 

分 类 号：S852.61[农业科学—基础兽医学]