程序性死亡分子1胞外区的表达及其抗血清对PCV2增殖的影响  

Expression of PD-1 extracellular domain and affection on PCV2 proliferation of rabbit serums against PD-1

作　　者：王林青[1,2] 徐朋丽 陈曦艋 李洪炫 陈红英[2] 赵丽[3] WANG Linqing;XU Pengli;CHEN Ximeng;LI Hongxuan;CHEN Hongying;ZHAO Li(Zhengzhou Key Laboratory of Molecular Biology,Zhengzhou Normal University,Zhengzhou 450044;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046;College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046)

机构地区：[1]郑州师范学院分子生物学郑州市重点实验室,郑州450044 [2]河南农业大学动物医学院,郑州450046 [3]河南牧业经济学院动物医药学院,郑州450046

出　　处：《安徽农业大学学报》2022年第3期443-448,共6页Journal of Anhui Agricultural University

基　　金：中原千人计划项目(204200510015);河南省高校科技创新人才支持计划(21HASTIT039);河南省高等学校青年骨干教师培养计划(2018GGJS161);河南牧业经济学院博士启动资金(2020HNUAHEDF010)共同资助。

摘　　要：为研究猪的程序性死亡分子1(PD-1)胞外区参与猪圆环病毒2型(porcine circovirus type 2,PCV2)感染的免疫调节功能,PCR扩增健康猪舌组织中PD-1胞外区,构建重组表达质粒p ET-28a-PD-1,转化至Rosetta(DE3)。经IPTG诱导表达后,采用SDS-PAGE和Western Blot检测重组PD-1胞外段蛋白ex PD-1表达情况。结果显示,重组菌p ET-28a-PD1/DE3,在37℃、0.5 mmol·L-1 IPTG诱导8 h时,ex PD-1蛋白的表达量最高,大小约为20 kDa,且以包涵体形式表达。然后将经纯化和复性获得的ex PD-1辅以佐剂免疫新西兰兔,制备兔抗ex PD-1血清,经Western Blot检测结果显示该抗血清具有较好的反应原性。用PCV2感染猪肾(Porcinekidney-15,PK-15)细胞,采用荧光定量PCR检测PD-1转录水平,结果显示PCV2感染PK-15细胞1~48 h时PD-1的m RNA转录水平均下调;用兔抗ex PD-1血清处理PK-15细胞后感染PCV2,PD-1的转录水平显著上调(P<0.01),且明显地抑制了PCV2的复制(P<0.01)。上述结果表明,在体外PCV2感染可引起PD-1转录水平下调,兔抗ex PD-1血清可通过封闭PD-1/PD-L1通路,抑制PCV2增殖。To investigate the immune regulation function of extracellular domain of programmed death molecule 1(PD-1) involved in porcine circovirus type 2(PCV2) infection, the extracellular domain gene of programmed death 1(PD-1) was amplified from pig tongue tissue, and the recombinant expression plasmid pet-28a-PD-1 was constructed and transformed into Escherichia coli Rosetta(DE3). After induced with IPTG, the expression of the recombinant PD-1 extracellular segment protein expd-1 was detected by SDS-PAGE and Western Blot. The results showed that the highest level of recombinant protein was observed when recombinant bacteria were induced by using 0.5 mmol·L-1 IPTG at 37 ℃ for 8 h. Western Blot showed that there was about 20 kDa specific brown red protein(inclusion body) band in the expected position. Then New Zealand rabbits were immunized with purified and renatured ex PD-1 supplemented with adjuvant to prepare rabbit anti-ex PD-1 serum. The results of fluorescence quantitative PCR showed that PD-1 was down-regulated in porcine kidney-15(PK-15) cells infected with PCV2 for 1-48 h;the transcription level of PD-1 was significantly up-regulated(P < 0.01), and the replication of PCV2 was inhibited significantly(P < 0.01) after the treatment of PK-15 cells with rabbit anti-PD-1 sera. The experiment results showed that PCV2 infection can cause the down regulation of PD-1 transcription level in vitro, and anti-exPD-1serum can inhibit the proliferation of PCV2 by blocking PD-1/PD-L1 pathway.

关 键 词：程序性死亡分子1 表达 兔抗PD-1血清 PCV2 

分 类 号：S858.285.53[农业科学—临床兽医学]