摘要
为了鉴定猪圆环病毒2型(PCV2)侵染细胞建立感染的重要宿主因子,本文分析在PK-15细胞中猪淋巴细胞特异性酪氨酸激酶Lck表达及活性对PCV2感染复制的影响,确定Lck是调控PCV2感染复制的新靶点。首先,本研究基于猪Lck结构特性和序列保守性分析,选取Lck SH3结构域为免疫原基因序列,利用原核表达目的蛋白制备小鼠抗猪Lck的多克隆抗体。然后,通过Western blot检测PCV2感染PK-15和3D4/21细胞后对Lck表达及活性的影响。最后,在PK-15细胞中利用瞬时转染过表达或Lck特异性抑制剂(A770041),通过Western blot和荧光定量PCR(RT-qPCR)检测猪Lck表达和磷酸化对PCV2复制的影响。结果显示,成功制备了鼠源性抗猪Lck的多克隆抗体。在PCV2感染PK-15和3D4/21细胞早期阶段,Lck表达水平未见明显变化,但Lck磷酸化水平显著上调。过表达Lck可显著上调PCV2 Cap蛋白表达及病毒拷贝数,而采用A770041抑制Lck磷酸化水平后,可显著下调PCV2 Cap蛋白表达、病毒拷贝数和病毒滴度,这些结果表明Lck正调控PCV2的感染复制,为发现PCV2感染的新分子机制奠定重要基础。
In order to identify important host factors in cell entry and establishment of infection of porcine circovirus type 2(PCV2),the effects of porcine lymphocyte specific tyrosine kinase Lck expression and activity on PCV2 infection replication were analyzed,and Lck was identified as a new target for the regulation of PCV2 replication.In this study,based on analysis of the structural characteristics and sequence conservation of porcine Lck,the Lck SH3 domain was selected as the immunogen gene sequence,and the polyclonal antibody against porcine Lck was prepared in mice using the protein expressed the E.coli expression system.Then,the effect of PCV2 infection on the expression and activity of Lck in PK-15 and 3D4/21 cells was detected using Western blot.Finally,the effects of porcine Lck expression and phosphorylation on PCV2 replication in PK-15 cells transiently transfected with overexpressed vector or treated with Lck specific inhibitor(A770041)were detected by Western blot and RT-qPCR,respectively.The results showed that mouse derived polyclonal antibodies against pig Lck were successfully prepared.There was no significant change in the expression level of Lck,but the phosphorylation level of Lck was significantly up-regulated in the early stage of PCV2 infection in PK-15 and 3D4/21 cells.Overexpression of Lck significantly increased PCV2 Cap protein expression and viral copy number,while inhibition of Lck phosphorylation level with A770041 could significantly downregulate the expression of PCV2 Cap protein,viral copy number and virus titer.These results indicated that Lck positively regulated PCV2 replication,which lays an important foundation for exploring the new molecular mechanism of PCV2 infection.
作者
刘伟姣
何庆
蒋一凡
曹思雨
张时瑞
吕佳璐
龙娉
杨凌宸
周川
王乃东
LIU Weijiao;HE Qing;JIANG Yifan;CAO Siyu;ZHANG Shirui;L Jialu;LONG Ping;YANG Lingchen;ZHOU Chuan;WANG Naidong(College of Veterinary Medicine,Hunan Agricultural University,Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines,R&D Center for Animal Reverse Vaccinology of Hunan Province,Changsha 410128,China;College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第12期5751-5761,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
湖南省自然科学基金面上项目(2022JJ30298)
湖南省技术攻关“揭榜挂帅”项目(2021NK1030)
湖南省大学生创新创业训练计划一般项目(S202310537036)。
