| 摘要: |
| 创伤弧菌是一种海洋环境中的重要病原菌。细菌脂蛋白是一类细菌重要的具有不同生理功能的膜蛋白,以大肠杆菌为代表的经典革兰氏阴性菌脂蛋白分泌规律遵循“+2规则”。为探究海洋环境中革兰氏阴性菌的细菌脂蛋白分泌机制是否有别于其他环境中的革兰氏阴性菌,本研究克隆并表达了创伤弧菌的MFP4脂蛋白,该脂蛋白N端成熟肽段第二位(+2位)氨基酸为甘氨酸,根据“+2规则”理论上应定位于细胞外膜。实验利用细菌外膜小体分离鉴定法,在表达MFP4重组脂蛋白的创伤弧菌中对其进行细胞定位后发现,MFP4生物合成后定位于细胞内膜。实验进一步构建了MFP4蛋白N端序列与红色荧光蛋白RFP的融合脂蛋白基因MFP4 tether-RFP并在创伤弧菌中表达,细胞定位结果发现该重组融合脂蛋白仍然定位于细胞内膜,说明MFP4成熟脂蛋白的N端携带内膜细胞定位的信息。实验同时构建并表达了外膜脂蛋白LptE的N端序列与RFP的融合重组脂蛋白LptE tether-RFP,该蛋白具有与MFP4相同的N端+2位氨基酸(Gly+2),与MFP4 tether-RFP融合脂蛋白仅有(+3 —+10位)8个氨基酸序列的差异。实验发现该融合脂蛋白定位于创伤弧菌细胞外膜,而它Gly+2→Asp的突变型蛋白则定位于细胞内膜,说明创伤弧菌脂蛋白+2位的Gly不是内膜滞留信号。研究数据表明,海洋环境中创伤弧菌脂蛋白MFP4的分泌及定位有其独特的规律,其细胞内膜定位信息由成熟脂蛋白N端+2位氨基酸以后的短肽序列决定。 |
| 关键词: 创伤弧菌 脂蛋白 分泌信号 细胞定位 |
| DOI：10.11759/hykx20220406002 |
| 分类号:S917.1 |
| 基金项目:国家自然科学基金面上项目(31372564);山东省自然科学基金面上项目(ZR2020MC197);山东省重点研发计划(公益类专项)(2017NC210001) |
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| Cloning, expression, and cellular localization of lipoprotein MFP4 in Vibrio vulnificus |
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Zhang Meng-lin1, MA Hong-shu2, ZHU Qing-song1, LI Ke1, WANG Wen-qi1, CHEN Shi-yong1
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1.School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China;2.Laboratory Management Center, Qingdao Agricultural University, Qingdao 266109, China
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| Abstract: |
| Vibrio vulnificus is a significant pathogen in the marine environment. The cellular localization and specific inner membrane retention signal of the predicted MFP4 lipoprotein were determined in the V. vulnificus genome to explore the secretion and cellular localization mechanism of lipoproteins. The mfp4 gene was cloned and constructed into a broad host overexpressing plasmid, and V. vulnificus overexpressed the MFP4 recombinant lipoprotein by bacterial conjugation. The recombinant lipoprotein MFP4 was further localized in the cell membrane by isolating the bacterial outer membrane vesicles. A fusion lipoprotein gene composed of the MFP4 N-terminal sequence and the red fluorescent protein (RFP) was constructed and expressed in V. vulnificus to confirm whether cellular localization of the MFP4 protein could be determined by the N-terminal disordered region of the mature lipoprotein. The results of cell localization revealed that the recombinant fusion lipoprotein was located in the inner membrane. Another fusion recombinant lipoprotein composed of the N-terminal region of the outer membrane lipoprotein LptE and RFP, which has the same N-terminal + 2 amino acids (Gly+2) and a difference of only (+3–+10) 8 amino acids, was constructed and expressed in V. vulnificus. That lipoprotein was located in the outer membrane, while the mutant with Gly+2 to Asp was located in the inner membrane. This result indicates that Gly at position +2 of the V. vulnificus lipoprotein was not a retention signal. These experimental data show that the secretion and localization of V. vulnificus lipoprotein MFP4 have unique rules, and its inner membrane localization was determined by the short peptide sequence after the N-terminal + 2 amino acids of the mature lipoprotein. |
| Key words: Vibrio vulnificus lipoprotein secretion signals cellular localization |
