鸡Rab7b在细胞晚期内吞体定位和结合恒定链的分子特征

doi:10.11843/j.issn.0366-6964.2020.12.024

摘要/Abstract

摘要： Rab蛋白是介导胞内活性蛋白转运的分子家族。恒定链（invariant chain，Ii）具有协助抗原肽转运、B细胞成熟和作为细胞因子的受体等免疫学功能。前期研究发现，鸡（Gallus gallus）Rab5a在细胞早期内吞体中与Ii结合，但不清楚是否还有其他转运蛋白有该特性。基于Rab7b分子定位于晚期内吞体，并与胞内分子转运相关，本研究探索了鸡Rab7b与Ii结合的分子结构特征。首先，用自行设计的引物扩增了鸡和小鼠（Mus musculus）Rab7b基因，并进行了氨基酸同源性比对分析；构建了含鸡Rab7b及其67位氨基酸突变体Rab7bQ67L的原核和真核重组质粒。其次，将含红色荧光蛋白和目的基因的重组质粒转染鼠巨噬细胞系Raw264.7，培养后用绿色荧光素（FITC）标记的鼠抗晚期内吞体蛋白1（LAMP1）抗体染色，观察目的蛋白在内吞体的定位。进一步将鸡Rab7b和Rab7bQ67L分别与Ii共转染293T细胞，观察它们与Ii的共定位。最后用拉下法和免疫印迹检测鸡Rab7b及Rab7bQ67L与Ii的结合。结果表明，所克隆获得的鸡Rab7b基因与预期大小一致，其开放阅读框为624 bp，编码208个氨基酸。鸡和鼠的Rab7b蛋白质结构相似性为74%。尽管鸡Rab7b和突变体Rab7bQ67L均能结合Ii，但只有Rab7b可以定位于晚期内吞体，而突变体却改变了该定位特性。综上所述，鸡Rab7b与Ii不仅在晚期内吞体共定位，而且还能互相结合；鸡Rab7b的第67位氨基酸影响其在细胞内定位而不影响与Ii结合。综上表明，Rab分子参与了Ii在细胞内细胞器的转运，为进一步研究Ii在细胞内的转运机制提供了新的途径。

关键词: Ii, 晚期内吞体, Rab7b, 突变, 共定位

Abstract: Rab is a family of molecules that mediate the transport of active proteins in cells. The invariant chain (Ii) has the immunological functions of assisting antigen peptide transport, B cell maturation, and acting as the receptor of cytokines. Previous studies showed that Rab5a of chicken (Gallus gallus) is bound to Ii in the early endocytosis, but it is not clear whether there are other Rab molecules with this property. Based on the localization of Rab7b in late endocytosis and its relationship with intracellular molecular transport, this study explored the molecular structure of Rab7b binding to Ii. First, Rab7b genes of chicken and mouse (Mus musculus) were amplified with self-designed primers, and the amino acid homology was analyzed; the prokaryotic and eukaryotic recombinant plasmids containing Rab7b and its 67 amino acid mutant Rab7bQ67L were constructed. Secondly, the recombinant plasmid containing red fluorescent protein and target gene was transfected into mouse macrophage line Raw264.7. After culture, the recombinant plasmid was stained with FITC labeled mouse anti late endosomal protein 1 (LAMP1) antibody to observe the localization of the target protein. Furthermore, chicken Rab7b and Rab7bQ67L were co-transfected with Ii to observe their co-localization with Ii. Finally, the binding of Rab7b and its mutants to Ii were detected by pull-down technique and Western blotting. The results showed that the Rab7b gene was the same size as expected, and its open reading frame was 624 bp, encoding 208 amino acids. The homology of Rab7b protein structure between chicken and mouse was 74%. Although both Rab7b and Rab7bQ67L could bind to Ii, it was Rab7b rather than Rab7bQ67L could locate in the late endocytosis. In conclusion, Rab7b and Ii were not only co-located in the late endocytosis, but also combined with each other; the 67th amino acid of Rab7b affected its location in cells but not with Ii. These results suggest that Rab molecules are involved in the transport of Ii in intracellular organelles, which provides a new way to further study the mechanism of Ii transport in cells.

Key words: Ii, late endocytosis, Rab7b, mutation, co-localization

中图分类号:

S852.4

陈芳芳, 谭红黎, 钱艳红, 桂亚萍, 于凤梅, 查丽莎. 鸡Rab7b在细胞晚期内吞体定位和结合恒定链的分子特征[J]. 畜牧兽医学报, 2020, 51(12): 3151-3159.

CHEN Fangfang, TAN Hongli, QIAN Yanhong, GUI Yaping, YU Fengmei, ZHA Lisha. Molecular Characteristics of Chicken Rab7b in Localization and Binding Invariant Chain in Late Cell Endocytosis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3151-3159.

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