福氏志贺菌5a型M90T株GAPDH蛋白多克隆抗体的制备和鉴定  

Preparation and identification of the polyclonal antibody against GAPDH protein from Shigella flexneri M90T

作　　者：王羽[1,2] 廖翔[2] 岳俊杰[2] 周围[2] 梁龙[2] 呼和巴特尔[1] 

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

出　　处：《细胞与分子免疫学杂志》2014年第4期407-410,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30970122)

摘　　要：目的制备高效的福氏志贺菌5a M90T3-磷酸甘油醛脱氢酶(GAPDH)的多克隆抗体,并对其特异性进行评价。方法提取M90T的全基因组,采用PCR法扩增M90T中的基因gapA克隆到原核表达载体pET-24中,重组质粒转入到大肠杆菌BL21(DE3)中进行诱导表达,条件优化后纯化GAPDH-His融合蛋白。以纯化的融合蛋白作为抗原免疫BALB/c小鼠,制备该蛋白的多克隆抗体。Western blot法、ELISA鉴定获得的抗血清。结果成功构建了原核表达载体pET24a-gapA,通过一系列诱导条件的优化,确定可溶性条件为30℃、1 mmol/L IPTG诱导2 h。用纯化的融合蛋白GAPDH-His作为抗原免疫小鼠制备多克隆抗体,Western blot法、ELISA证明多克隆抗体制备成功。结论成功制备了福氏志贺菌5a M90T特异性的小鼠多克隆抗体。Objective To prepare mouse polyclonal antibodies against Shigella flexneri Mg0T glycereldehyde-3- phosphate dehydrogenase （GAPDH）, and verify its titer and specificity. Methods The gapA gene of Shige/la flexneri Mg0T was amplified by PCR from extracted genomic DNA and inserted into expression vector pET24a. The plasmid pET24a-gapA was transformed into E. coil BI_2] （DE.3） and induced to express the tagged protein GAPDH-His. After condition optimization, the protein was purified with Ni SepharoseTM 6 Fast Flow. The polyclonal antibodies were prepared by immunizing the mice with the purified recombinant protein and then harvesting mouse sera. The specificity and efficiency of antibodies in sera were analyzed by Western blotting and ELISA against whole cell proteins of Shigella flexneri Mg0T. Results The recombinant plasmid pET24a-gapA was successfully constructed. The soluble expression of the tagged protein GAPDH-His was achieved with 2-hour induction of l mmoVL IPTG at 30~（3. Western blotting and ELISA demonstrated that the polyclonal antibody harvested from the mice immunized with GAPDH-His was specific and efficient against Shigella flexneri Mg0T GAPDH. Conclusion The mouse polyclonal antibodies against the Shige/la f/exneri Mg0T GAPDH protein were successfully prepared.

关 键 词：福氏志贺菌5aM90T 3-磷酸甘油醛脱氢酶 多克隆抗体 

分 类 号：R392.33[医药卫生—免疫学] R378.25[医药卫生—基础医学]