摘要
目的:制备兔抗人PON2(paraoxonase-2)的多克隆抗体并进行初步鉴定。方法:生物信息学方法分析人PON2蛋白序列,选取人兔同源性较低,亲水性及免疫原性较强的片段,通过大肠杆菌原核表达系统进行重组表达,获得GST-PON2融合蛋白用于免疫新西兰大耳白兔获得多克隆抗体,通过XX方法分离纯化得到的抗PON2多克隆抗体,用West-ernblot、间接免疫荧光对其进行特异性及灵敏度鉴定。结果:成功获得了高表达的相对分子质量(Mr)为46000的PON2重组融合蛋白;Westernblot鉴定结果显示此多克隆抗体可特异识别肝脏总蛋白、HeLa细胞及U937细胞中Mr为39000的天然PON2蛋白;间接免疫荧光结果显示此多克隆抗体所识别的蛋白定位于SY5Y细胞胞质中。结论:抗人PON2的多克隆抗体特异识别肝总蛋白、HeLa细胞及U937细胞中的天然蛋白,可用于PON2的研究及临床检测。
AIM： To preparation and characterize the rabbit polyclonal antibodies against human PON2 （paraoxonase-2）. METHODS： A fragment of human PON2 gene which was of low homology with rabbits but of highe hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times. The specificity and sensitivity of the anti-human PON2 polyclonal antibodies were detected by Western blot and indirect immunofluorescence. RESULTS： The GSTPON2 fusion protein was highly expressed in Ecoli with a molecular weight of 46 kDa. Westem blot analysis proved the rabbit polyclonal antibodeies could specifically recognize 39 kDa native PON2 protein expressed in several cells and tissues, such as HeLa cells, U937 cells, and human liver tissue. Indirect immunofluorescence analysis confirmed that PON2 protein was located in the cytoplasm of SYSY cells. CONCLUSION： The rabbit polyclonal antibodies against human PON2 can specifically recognize natural protein expressed in human cells and tissues, Which can be used for further study and clinical detection of human PON2.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第7期699-702,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划(863)资助项目(2006AA02A311)
