Study on Vero Cell Adhesion Related Proteins Based on TMT Proteomics
ZHANG Xiao-Wen1,2, YANG Xiao-Li1,2, BI Dong-Lin1,2, YANG Dong-Liang1,2, LIU Fang-Cheng1,2, LING Shi-Xin1, BAI Jia-Lin1,2,*, LI Qiong-Yi1,2, ZHANG De-Rong1
1 Gansu Innovation Center for Animal Cell/Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China; 2 College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China
Abstract 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 integrin β 2 (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 levels of ITGβ2 was most significantly up-regulated (P<0.01). Western blot analysis showed that the protein expression of SDC4 in Sus_Vero was significantly decreased compared with that in Adh_Vero. The trend of protein expression was consistent with quantitative proteomics. The findings preliminarily revealed the adhesion mechanism of Vero cells and provided an effective target proteins for the further construction of Sus_Vero culture using genetic engineering technology.
ZHANG Xiao-Wen,YANG Xiao-Li,BI Dong-Lin, et al. Study on Vero Cell Adhesion Related Proteins Based on TMT Proteomics[J]. 农业生物技术学报, 2023, 31(10): 2150-2162.
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