摘要
旨在探究利用蛋白免疫印迹手段测定施氏假单胞菌胞内关键酶NapA和NapB表达量的可行性。选取多肽片段合成免疫抗原,并用其免疫兔子获得相应的多克隆抗体。ELISA检测第三次免疫后两周的兔抗血清的效价,并将最终免疫产生的抗体用于施氏假单胞菌样品的蛋白免疫印迹分析。制备的多肽抗原具有良好的免疫原性,NapB免疫原效果甚佳,血清效价大于1∶160000,NapA的效价也可达到1∶14000。免疫印迹试验结果的发光图像中内参条带清晰整齐,在目标蛋白条带区域也可以看到明显反应带。本研究制备出的抗体可用于施氏假单胞菌胞内NapA和NapB蛋白的免疫印迹分析,后续还可以改变实验条件通过内参蛋白进行半定量。
The purpose of the study is to explore the feasibility of key enzyme(NapA/NapB)assay in Pseudomonas stutzeri using Western Blotting.[Method]Peptides were designed and synthesized to immune rabbits to obtain polyclonal antibodies of NapA and NapB,respectively.The antiserum titer was tested by ELISA and the final antibodies were used in Western Blotting detection towards cultured strains.The customized antigens showed good immunogenicity,with a tier over 1∶160000 of NapB and a tier up to 1∶14000 of NapA.The loading control band was clear and consistent in both protein extracts from the samples.And the immuno detected bands were obviously visible in target region as well.Our customized antibodies could be applied well to detect the protein content of NapA and NapB in Pseudomonas stutzeri through Western Blotting and semi-quantification analysis could be realized in subsequent experiments.
作者
周萌
柴晓利
Zhou Meng;Chai Xiaoli(College of Environmental Science and Technology,Tongji University,Shanghai 200092,China)
出处
《山东化工》
CAS
2019年第20期25-28,31,共5页
Shandong Chemical Industry
基金
重污染河流负荷削减与污染控制技术集成与示范(2017ZX07202002)
