基于TMT蛋白质组学对Vero细胞粘附相关蛋白的研究  

Study on Vero Cell Adhesion Related Proteins Based on TMT Proteomics

作　　者：张潇文 杨晓莉 毕冬琳 杨东亮 刘方程 令世鑫[1] 柏家林[1,2] 李琼毅 张德荣[1] ZHANG Xiao-Wen;YANG Xiao-Li;BI Dong-Lin;YANG Dong-Liang;LIU Fang-Cheng;LING Shi-Xin;BAI Jia-Lin;LI Qiong-Yi;ZHANG De-Rong(Gansu Innovation Center for Animal Cell/Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学生物医学研究中心/甘肃省动物细胞技术创新中心,兰州730030 [2]西北民族大学生命科学与工程学院,兰州730030

出　　处：《农业生物技术学报》2023年第10期2150-2162,共13页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31860696);兰州市人才创业创新项目(2020-RC85);甘肃省自然基金(21JR11RA018)。

摘　　要：细胞-细胞及细胞-细胞外基质的粘附取决于细胞特有的粘附蛋白及其相关调节信号。非洲绿猴肾脏上皮细胞(African green monkey kidney epithelial cells,Vero)是流感病毒(Influenza virus)、新型冠状病毒(Novel coronavirus)等多种病毒繁殖、纯化和疫苗生产的优良细胞株,为进一步探寻与Vero细胞粘附相关的蛋白,继而有效提升大规模高密度悬浮培养,本研究利用基因修饰及无血清培养基细胞技术成功构建悬浮型Vero细胞,并通过串联质谱标签(tandem mass tag,TMT)定量蛋白质组学技术对贴壁培养Vero细胞(adherent culture Vero,Adh_Vero)和悬浮培养Vero细胞(suspension culture Vero,Sus_Vero)中与细胞粘附性能相关的差异表达蛋白进行分析,以期为与悬浮培养Vero细胞粘附相关的候选靶基因筛选提供依据。结果表明,与Adh_Vero细胞相比,Sus_Vero细胞中表达差异显著的蛋白质有190个,其中上调表达蛋白116个,下调表达蛋白74个。通过GO功能注释和KEGG通路富集筛选获得与粘附相关的差异表达蛋白6个,其中5个上调表达蛋白分别是多配体蛋白聚糖-4(syndecan 4,SDC4)、肌动蛋白调节器(enabled homolog,ENAH)、肌球蛋白轻链12β(myosin light chain 12β,MYL12β)、肌球蛋白轻链激酶(myosin light chain kinas,MYLK)和封闭蛋白(occludin,OCLN),1种下调表达蛋白为整合素β2蛋白(integrinβ2,ITGβ2)。qPCR检测表明,Sus_Vero中SDC4、OCLN、MYL12β、MYLK以及ENAH的m RNA水平极显著下调(P<0.01),ITGβ2的m RNA水平极显著上调(P<0.01)。Western blot检测表明,Sus_Vero中SDC4蛋白表达量也比贴壁培养细胞中显著减少,与定量蛋白质组学蛋白质表达趋势一致。本研究结果初步揭示了Vero细胞的粘附机制,为进一步运用基因工程技术构建Sus_Vero提供了有效候选靶蛋白。Cell-cell and cell-extracellular matrix adhesion depends on cell-specific adhesion proteins and their associated regulatory signals.African green monkey kidney epithelial cells(Vero)are excellent cell lines for the proliferation,purification and vaccine production of multiple important viruses such as Influenza virus,Novel coronavirus.During in vitro culture,Vero cells usually grow in the medium containing serum with two-dimensional single-cell layer attached to the wall of petri dishes.Biomedical Research Center of Northwest Minzu University obtained Vero cell lines that could be suspended for culture by using culture method of gradually lowering serumin the medium,but the cell density was low.It is difficult to achieve large-scale high density suspension culture.Gene modification techniques and serum-free culture-medium cell screening techniques have been successfully applied to the construction of suspension cell lines.To construct suspension Vero cells using genetic engineering methods,it is necessary to screen proteins related to their adhesion function and understand how these proteins affect cell adhesion.In this study,tandem mass tag(TMT)quantitative proteomics was used to analyze the types and amounts of differential expression proteins associated with cell adhesion Vero cells(Adh_Vero)and suspension Vero cells(Sus_Vero).Compared with Adh_Vero group,the results showed that there were 190 differential expression proteins in Sus_Vero cells,including 116 up-regulated proteins and 74 down-regulated proteins.Six differentially expressed proteins related to cell adhesion were obtained by GO functional annotation and KEGG pathway enrichment screening.The up-regulated 5 proteins were syndecan 4(SDC4),enabled homolog(ENAH),myosin light chain 12β(MYL12β),myosin light chain kinas(MYLK),occludin(OCLN),and 1 protein of integrinβ2(ITGβ2)was down-regulated.Real-time quantitative PCR(qPCR)showed that the mRNA levels of SDC4,OCLN,MYL12β,MYLK and ENAH were most significantly down-regulated(P<0.01),while the mRNA leve

关 键 词：非洲绿猴肾脏上皮细胞(Vero) 细胞悬浮培养 蛋白质组学 细胞粘附 多配体蛋白聚糖-4(SDC4) 

分 类 号：S85[农业科学—兽医学] Q291[农业科学—畜牧兽医]