猪PD-1胞外区基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Porcine PD-1 Extracellular Region Gene

作　　者：杨媛[1,2] 朱艳平[1] 岳锋[1] 孙国鹏[1] 张艳芳[1] 银梅[2] 王选年[1] 

机构地区：[1]新乡学院生物技术研究中心,生命科学与技术系,河南新乡453003 [2]河南科技学院动物科学学院,河南新乡453003

出　　处：《动物医学进展》2015年第9期55-59,共5页Progress In Veterinary Medicine

基　　金：河南省基础与前沿技术研究(122300410150);国家自然科学基金项目(31272539,31201877);河南省教育厅科学技术研究重点项目(13A230837)

摘　　要：为研究PD-1/PD-L1通路在猪免疫抑制性疾病中的作用,根据猪的PD-1的基因序列,设计扩增其胞外区的引物,从猪PBMC基因组中通过PCR扩增获得猪PD-1胞外区基因片段,测序正确后克隆至原核表达载体pET-32a(+),构建重组原核表达质粒pET32-PD1,转化大肠埃希菌DH5α。挑选可疑菌落鉴定正确后转至Rosetta(DE3)进行诱导表达,表达产物进行SDS-PAGE和Western blot分析。结果表明,在37℃、0.5mmol/L IPTG条件下诱导获得33ku的PD-1胞外区融合蛋白,能够被其多抗血清、His标签抗体和人PD-1抗体所识别。本研究为制备其单克隆抗体和研究PD-1/PD-L1在猪免疫抑制性疾病中的作用提供了材料。In order to study the role of porcine PIN1/PD-L1 pathways in immunosuppressive disease, the primers were designed according to the extracellular region of porcine PD-1 sequence in GenBank, and the porcine PD-1 ex- tracellular region gene was cloned from porcine PBMC. The PCR products were determined by cloning and sequen- cing, and then directionally cloned into pET32a（-k）. The recombinant plasmid in E. coli Rosetta （DE3） was in- duced with IPTG, and the expression products were detected by SDS-PAGE and Western blot. SDS-PAGE analy- sis showed that we obtained the recombinant proteins at 37 ~C, 0.5 mmol/L IPTG, and its molecular weight is 33 ku. Western blot revealed that the expressed products were reacted by anti-porcine PD-1 antibody, His-tag antibody and anti-human PD-1 monoclonal antibody. These results indicated that the recombinant prokaryotic expression protein of porcine PD-1 was properly expressed. The protein has a good antigenecity and reactogenicity. It is useful to prepared the monoclonal antibodies and study the PD-1/PD-L1 function in immunosuppressive diseases in pigs.

关 键 词：猪PD-1 胞外区 克隆 原核表达 

分 类 号：Q786[生物学—分子生物学] S858.28[农业科学—临床兽医学]