志贺菌福氏5a M90T毒力蛋白Apyrase的表达、纯化及多克隆抗体制备  

Expression and purification of Apyrase from shigella flexneri M90T and preparation mice polyclonal antibody against Apyrase

作　　者：王羽[1] 周围 廖翔 高原 戴红梅 岳俊杰 梁龙 呼和巴特尔[1] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]军事医学科学研究院生物工程研究所,北京100071

出　　处：《中国免疫学杂志》2013年第10期1080-1084,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(30970122)资助项目

摘　　要：目的:克隆编码志贺菌福氏5a M90T毒力蛋白Apyrase的基因phoN2并实现表达,纯化后免疫BALB/c小鼠制备鼠抗Apyrase的多克隆抗体,并对多克隆抗体进行评价。方法:通过PCR扩增的志贺菌5a M90T中的基因phoN2克隆至表达载体pET24a中,将重组质粒转化到E.coli BL21(DE3)中,筛选阳性克隆,进行IPTG诱导表达并纯化Apyrase-HIS融合蛋白。以纯化的融合蛋白作为抗原免疫BALB/c小鼠,制备该蛋白的多抗。Western blot、ELISA、免疫荧光鉴定获得的抗血清。结果:成功构建了原核表达载体pET24a-Apyrase,经诱导表达出相对分子量为28 kD的融合蛋白,用纯化的融合蛋白作为抗原免疫小鼠制备的多克隆抗体,通过Western blot、ELISA、免疫荧光法证明多克隆抗体制备成功。结论:成功制备了志贺菌福氏5a特异性的鼠多克隆抗体,为进一步研究Apyrase蛋白在志贺菌福氏5a M90T中的表达、定位及志贺菌福氏5a M90T的快速检测奠定了基础。Objective：To fulfill recombinant expression and purification of protein Apyrase, and prepare the polyelonal anti- bodies of mice anti-Apyrase. Methods ： The pnoN2 gene of shigella.flexneri M90T was amplified by PCR and inserted into expression vector pET24a. The resultant recombinant expression vector pET24a-pno2 was transformed into E. eoli BI21 （DE3） and induced with IPTG, then recombinant protein purified with Nl-eolumn. The polyelonal antibody was prepared from the mice immunized with purified recombinant protein and then analyzed by Western blot, ELISA and Immunofluoreseent staining. Results： The recombinant plasmid pET24a-Apyrase was successfully expressed and purified. The polyclonal antibody harvested from mice immunized with Apyrase-HIS were demonstrated by WB and ELISA being specific and efficient for detecting Apyrase. Conclusion： The polyclonal antibodies against the Apyrase protein were successfully prepared, which is good tool not only for studying expression, localization and function of Apyrase, but also for identifying shigeUaflexneri efficiently and specifically.

关 键 词：志贺菌福氏5a M90T APYRASE 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]