稳定表达IL-10的PK-15细胞的构建及其在PCV2增殖中的应用  

Construction of PK-15 Cells Stably Expressing IL-10 and Its Application in PCV2 Proliferation

作　　者：张敬寒 李枝兰 韩佃刚 何帅 刘金华[3] 吕念词 邹丰才 柴俊 张以芳 ZHANG Jinghan;LI Zhilan;HAN Diangang;HE Shuai;LIU Jinhua;LYU Nianci;ZOU Fengcai;CHAI Jun;ZHANG Yifang(College of Veterinary Medicine, Yunnan Agricultural University,Kunming 650201, China;Kunming Customs Technology Centre,Kunming 650051,China;College of Veterinary Medicine, China Agricultural University,Beijing 100193,China)

机构地区：[1]云南农业大学动物医学院,昆明650201 [2]昆明海关技术中心,昆明650051 [3]中国农业大学动物医学院,北京100193

出　　处：《中国畜牧兽医》2022年第3期1085-1095,共11页China Animal Husbandry & Veterinary Medicine

基　　金：云南省专家工作站项目(202005AF150041);国家重点研发计划专项(2017YFD0500105)。

摘　　要：【目的】研究白细胞介素-10(IL-10)对猪圆环病毒2型(Porcine circovirus type 2,PCV2)复制的影响,筛选PCV2高感染性细胞系和提高PCV2病毒滴度,为后续疫苗的研发及IL-10在PCV2感染中的作用研究提供参考。【方法】利用PCR技术扩增猪IL-10基因,将目的基因与慢病毒表达载体(pCDH-CMV-MCS-EF1-GFP+Puro)进行连接,获得重组质粒pCDH-CMV-IL-10,将其与包装质粒psPAX2和pMD2.G共转染293T细胞进行慢病毒包装。用收集的慢病毒液感染PK-15细胞,经嘌呤霉素筛选后得到细胞株PK-15-IL-10,对照组细胞分别命名为PK-15-pCDH和PK-15。PCV2感染PK-15-IL-10、PK-15-pCDH和PK-15细胞株后,在24、48和72 h分别收集细胞液,利用CCK-8检测细胞活力。利用实时荧光定量PCR和Western blotting检测IL-10基因的表达水平和PCV2的复制情况;利用间接免疫荧光试验(IFA)观察PCV2在细胞中的复制情况及测定PCV2的病毒滴度(TCID50)。【结果】试验成功构建了重组质粒pCDH-CMV-IL-10,将其与包装质粒psPAX2和pMD2.G共转染293T细胞后,48 h时细胞状态最好,荧光最强。分别收集共转染48和72 h的慢病毒液上清感染PK-15细胞,pCDH-CMV-IL-10组的荧光最强,将其在嘌呤霉素浓度为2.5μg/mL的完全培养基中继续培养,获得仍有绿色荧光的稳转细胞株。实时荧光定量PCR和Western blotting检测发现,IL-10基因在pCDH-IL-10细胞株中的表达量明显高于对照组PK-15-pCDH和PK-15,PCV2的拷贝数增加了4倍,复制能力增强,且将病毒稀释连续传3代后,PK-15-IL-10细胞中的PCV2极显著高于PK-15细胞(P<0.01)。细胞增殖试验表明,猪IL-10基因在细胞中过表达对细胞活力无明显影响;IFA结果表明,PK-15-IL-10细胞中的荧光比PK-15细胞更强,PCV2在PK-15-IL-10细胞中的TCID50在感染后48 h极显著高于PK-15细胞(P<0.01)。【结论】本研究成功构建了pCDH-CMV-IL-10的慢病毒表达载体,并利用其感染PK-15细胞,继续培养后筛选出过表达IL-10的PK-15-IL-10细胞�【Objective】This experiment was conducted to investigate the effects of interleukin-10(IL-10)on the replication of Porcine circovirus type 2(PCV2),screen high infectious PCV2 cell lines,and improve the virus titer of PCV2,and lay a foundation for the subsequent vaccine research and development and the study of the role of IL-10 in PCV2 infection.【Method】Porcine IL-10 gene was amplified by PCR,the target gene was linked to lentivirus expression vector(pCDH-CMV-MCS-EF1-GFP+Puro),and the recombinant plasmid pCDH-CMV-IL-10 was obtained.It was co-transfected with package plasmids psPAX2 and pMD2.G into 293T cells for lentivirus packaging.PK-15 cells were infected with the collected lentivirus supernatant and screened by puromycin to obtain PK-15-IL-10 cell lines.The control cells were named PK-15-pCDH and PK-15,respectively.After PCV2 was infecting PK-15-IL-10,PK-15-pCDH and PK-15 cell lines,cell fluid was collected at 24,48 and 72 h,respectively,and cell viability was detected by CCK-8.The expression of IL-10 gene and the replication of PCV2 were detected by Real-time quantitative PCR and Western blotting.Indirect immunofluorescence assay(IFA)was used to observe the replication of PCV2 in cells and determine the virus titer of PCV2(TCID50).【Result】The recombinant plasmid pCDH-CMV-IL-10 was successfully constructed.293T cells were co-transfected with the packaged plasmids psPAX2 and pMD2.G,and the cell status was the best and the fluorescence was the strongest at 48 h.PK-15 cells were co-transfected with lentivirus supernatant for 48 and 72 h,respectively.The fluorescence of pCDH-CMV-IL-10 group was the strongest,and they were cultured in complete medium with puromycin concentration of 2.5μg/mL to obtain stable cell lines with green fluorescence.Real-time quantitative PCR and Western blotting results showed that the expression of IL-10 gene in pCDH-IL-10 cell lines was significantly higher than that in control group PK-15-pCDH and PK-15.The copy number of PCV2 was increased by four times,and its replicatio

关 键 词：猪圆环病毒2型(PCV2) 白细胞介素-10(IL-10) 免疫抑制 慢病毒表达载体 稳定表达细胞系 

分 类 号：S852.659.2[农业科学—基础兽医学]