MFG-E8作为外泌体载体蛋白的作用与功能

doi:10.13560/j.cnki.biotech.bull.1985.2021-0814

摘要/Abstract

摘要：

构建MFG-E8全长和截短体的重组质粒,分析各截短体蛋白能否将目的蛋白带到外泌体上并确定其位置,以探讨MFG-E8各结构域功能及其作为外泌体上载体蛋白的效率。利用PCR扩增出带有信号肽的MFG-E8及其截短体基因,与EGFP基因连接,构建7种重组质粒。之后PEI介导瞬转HEK293F细胞进行表达,利用EGFP的荧光,通过Western blot和激光共聚焦显微镜检测蛋白表达情况及其在胞内的位置。同时提取转染细胞分泌的外泌体,通过Western blot和电镜检测重组蛋白在外泌体中的情况。最后将含有重组蛋白的外泌体转染293T细胞,荧光显微镜观察外泌体将重组蛋白带入受体细胞中的情况。结果表明,成功构建MFG-E8全长及截短体重组质粒并在HEK293F细胞中表达,各重组蛋白在胞内均有表达,其中EGF-L表达量最高,C2最低,细胞上清中只能检测到EGF-L、C1、EC1的表达。转染细胞分泌的外泌体只检测到MFG-E8、EC1和C1C2三种蛋白。含有这3种蛋白的外泌体转染HEK293T细胞,3组细胞均有荧光,其中MFG-E8-EGFP的外泌体效果最好。MFG-E8能够作为蛋白载体将目的蛋白带到外泌体膜上,其C1、C2结构域对该过程至关重要,EGF-L的存在可以促进蛋白表达。

关键词: 乳脂肪球表皮生长因子Ⅷ（MFG-E8）, 截短体, 外泌体, 载体蛋白, 结构域功能

Abstract:

The objectives of this work are to construct the recombinant plasmids of full-length and truncated MFG-E8（milk fat globule epidermal growth factor 8）,to analyze whether each truncated protein can bring the target protein to exosomes determine their positions,for exploring the function of each domain of MFG-E8 and the efficiency as carrier proteins in exosomes. Polymerase chain reaction（PCR）was used to amplify the MFG-E8 and its truncated genes with signal peptide and fused with EGFP gene to construct 7 recombinant plasmids. Then,the plasmids were transfected into HEK293F cells using PEI. Western blot and laser scanning confocal microscope（LSCM）were to detect the proteins expressions and intracellular locations of MFG-E8 and its truncated recombinant proteins,respectively. Meanwhile,the exosomes secreted by the transfected cells were extracted and the recombinant proteins in the exosomes were detected by Western blot and electron microscopic. Finally,the exosomes harbored recombinant proteins were transfected into HEK293T cells and the results of exosomes bringing the recombinant proteins into receptor cells were observed by fluorescence microscopy. Results showed that MFG-E8 and its truncated plasmids were constructed successfully and expressed in HEK293F cells. All recombinant proteins were expressed in the cells,of them the expression of EGF-L was the highest,while the lowest for C2. Only EGF-L,C1 and EC1 were detected in the cell supernatant. MFG-E8,EC1 and EC2 were detected in the exosomes secreted by the transfected cells. With these exosomes containing 3 proteins transfected into HEK293T cells,the fluorescence was observed in all three groups of cells,of which the exosome from MFG-E8-EGFP was the most effective. In conclusion,MFG-E8 can be used as a carrier to bring the proteins of interest to the exosome membrane,and its C1 and C2 domains are crucial to this process. The presence of EGF-L may promote protein expression.

Key words: milk fat globule-epidermal growth factor VIII（MFG-E8）, truncated protein, exosome, carrier protein, function of domain

黎智康, 刘晨雪璇, 谭楚敏, 熊盛, 谢秋玲. MFG-E8作为外泌体载体蛋白的作用与功能[J]. 生物技术通报, 2022, 38(4): 288-294.

LI Zhi-kang, LIU Chen-xue-xuan, TAN Chu-min, XIONG Sheng, XIE Qiu-ling. Roles and Functions of MFG-E8 as Carrier Proteins in Exosomes[J]. Biotechnology Bulletin, 2022, 38(4): 288-294.

图/表 6

图1 MFG-E8-EGFP及截短体结构及酶切鉴定凝胶电泳图 A：MFG-E8-EGFP及截短体结构重组质粒示意图;B：酶切鉴定质粒凝胶电泳图

Fig. 1 Structure of MFG-E8-EGFP and its truncated forms as well as gel electrophoresis plots for identification by enzyme digestion A：Schematic diagram of the recombinant plasmids of MFG-E8-EGFP and its truncated forms. B：Gel electrophoresis plots for identifying the recombinant plasmids by enzyme digestion

图2 重组质粒瞬转效率和表达情况的检测 A：流式检测重组质粒转染HEK293细胞的效率;B：WB检测目的蛋白在HEK293细胞中的表达;C：WB检测目的蛋白在细胞上清中的表达

Fig. 2 Detection of the transient efficiency and expression of the recombinant plasmids A：Detection of the transient efficiency of the recombinant plasmids in HEK293 cells by flow cytometry. B：Detection of target protein expression in HEK293 cells by Western blot. C：Detection of target protein expression in culture supernatant by Western blot

图3 激光共聚焦显微镜检测MFG-E8及其截短体在细胞中的定位

Fig. 3 Location detection of MFG-E8 and its truncated pr-oteins in HEK293 cells by laser confocal microscopy

图4 外泌体的电镜结构和粒径分析 A：外泌体的电镜结构,scale bar,100 nm;B：NTA检测外泌体的粒径分布

Fig. 4 Transmission electron micrograph and size distribu-tions of exosomes A：Transmission electron micrograph of exosomes,scale bar,100 nm. B：Size distributions of exosomes on NTA measurement

图5 EGFP在外泌体中存在情况的鉴定 A：外泌体中MFG-E8-EGFP及其截短体的WB检测;B：分别转染pCMV-MFG-E8-EGFP（b）、pCMV-EC1-EGFP（c）和pCMV-C1C2-EGFP（d）的HE-K293F的外泌体电镜图;Scale bars,100 nm

Fig. 5 Identification of EGFP in exosomes A：Western blot detection of MFG-E8-EGFP and its truncated ones in exosomes.B：Transmission electron microscopy images of exosomes derived from HEK293F cell transfected with pCMV-MFG-E8-EGFP（b）,pCMV-EC1-EGFP（c）or pCMV-C1C2-EGFP（d）. Scale bars,100 nm

图6 荧光显微镜观察外泌体转染效果

Fig. 6 Fluorescence images showing the effect of exosomes transfection

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