转位紧密黏附素受体细胞骨架偶联蛋白基因克隆、表达、抗体制备及应用  

Gene cloning,expression and polyclonal antibody preparation and application of translocated intimin receptor-cytoskeleton coupling protein

作　　者：杨琴[1] 曹军皓[1] 丁进亚[1] 黄前川[1] 

机构地区：[1]广州军区武汉总医院检验科,湖北武汉430070

出　　处：《细胞与分子免疫学杂志》2012年第12期1282-1285,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30670107)

摘　　要：目的:克隆转位紧密黏附素受体细胞骨架偶联蛋白(TccP)基因,表达纯化TccP,制备其多克隆抗体并研究该抗体在EHEC O157∶H7黏附宿主细胞模型中的应用。方法:从EHEC O157∶H7 Sakai菌株基因组中克隆得到tccP基因,构建重组表达质粒pET28a-TccP。将此质粒转化大肠杆菌BL21(DE3)后用异丙基-D-硫代半乳糖苷(IPTG)诱导表达。经纯化分离后,免疫新西兰大白兔,制备其多克隆抗体,并用ELISA法和Western blot法对其进行鉴定。使用兔抗TccP多克隆抗体对EHEC O57∶H7黏附宿主细胞模型中的TccP分子进行定位研究。结果:获得了TccP cDNA,与GenBank报道的序列一致,并成功构建了高效原核表达质粒pET28a-TccP;Western blot结果表明,经IPTG诱导,在大肠杆菌BL21(DE3)中表达了相对分子质量(Mr)约37 000的目的蛋白。表达产物N端氨基酸序列测序结果与GenBank公布的基因序列推定的氨基酸序列完全一致。所制备的兔抗TccP多克隆抗体经Western blot法测定可与Mr37 000目的蛋白发生特异性结合反应;免疫荧光检测发现TccP聚集在EHEC O157∶H7黏附处细胞膜的下方。结论:成功构建了pET28a-TccP载体,获得了高纯度的重组TccP及其特异性多克隆抗体。AIM： To prepare translocated intimin receptor-cytoskeleton coupling protein （TccP） of Enterohaemorrhagic Escherichia coil （EHEC） O157：H7 and its polyclonal antibody. METHODS： TccP was amplified from the genome of EHEC O157：H7 Sakai strain by PCR and used to construct the recombinant prokaryotic expression vector pET28a-TccP. The recombinant vector was transformed into E. coil BL21 （DE3） to express the protein in the bacteria under the induction of isopropy-D-thiogalactoside （IPTG）. After purification, the protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by ELISA and Western blotting for its sensitivity and specificity. The rabbit anti-TccP polyclonal antibody was then applied in the study on the localization of TccP within the host cells adhered by EHEC O157：H7. RESULTS： The sequence of TccP cDNA we amplified was the same as reported by GenBank. The recombinant prokaryotic expression vector pET28a-TccP was constructed successfully. Western blotting revealed that Mr of the target protein expressed in E. coil BL21 （ DE3 ） was 37 000 and the rabbit anti-TccP polyclonal antibody had a specific reaction with the target protein, which demonstrated that the recombinant protein and its polyclonal antibody were prepared successfully. Immunofluorescence detection using rabbit anti-TccP polyclonal antibody showed that TccP aggregated in the cell membrane of the host cell adhered by EHEC O157 ： H7.CONCLUSION： We successfully prepared the recombinant vector pET28a-TccP and the anti-TccP polyclonal antibody and applied the antibody to confirm the localization of TccP in EHEC O157：H7 adhesion host cells.

关 键 词：肠出血性大肠杆菌O157∶H7 转位紧密黏附素受体细胞骨架偶联蛋白 基因表达 多克隆抗体 

分 类 号：R378.21[医药卫生—病原生物学]