抗原血清学识别重组表达克隆技术的优化

背景:抗原血清学识别重组表达克隆技术的应用有许多报道,但存在假阳性较高、稳定性欠佳等诸多问题。目的:优化抗原血清学识别重组表达克隆技术,建立灵敏且稳定的技术体系。方法:利用抗原血清学识别重组表达克隆技术,比较血清的处理方法、异丙基-β-D硫代半乳糖苷的加入方式、不同的封闭条件及培养基厚度对显色结果的影响,观察噬菌斑显色深浅、背景处理及假阳性的产生。结果与结论:直接法预处理血清、异丙基-β-D硫代半乳糖苷浸泡硝酸纤维素膜并干燥结合脱脂奶粉进行封闭,显色效果最佳。此外选择适当的培养基厚度可有效消除边缘效应。提示血清预处理、异丙基-β-D硫代半乳糖苷的加入方式、不同封闭条件及培养基厚度等因素,在利用抗原血清学识别的重组表达克隆技术进行抗原筛选中起关键作用。

Optimization of serological identification of antigens by recombinant expression cloning

BACKGROUND:In recent years,there are many reports on serological identification of antigens by recombinant expression cloning(SEREX) technology.However,there are higher false positive and poor stability problems.OBJECTIVE:To establish a sensitive and stability system by optimizing the SEREX technology.METHODS:The SEREX technology was used to compare the serum pretreat methods,isopropyl-beta-D-thiogalactoside adding methods,and the effect of different blocking conditions and medium thickness on the results of the coloration,and the plaque stained depth or not,background clean or not and false positive produce or not were observed.RESULTS AND CONCLUSION:The best result was obtained using following conditions:the serum was incubated directly;nitrocellulose membrane was immersed in isopropyl-beta-D-thiogalactoside and dried in air and then blocked with skimmed-milk;the thickness of the medium could efficiently eliminate the brim effect.It suggests that the serum pretreat methods,isopropyl-beta-D-thiogalactoside adding methods,as well as different blocking conditions and medium thickness play a key role in the selection of antigen by using SEREX technology.