鸡恒定链分子跨膜区2个氨基酸残基在形成MHCⅡ-Ii复合物中的作用

摘要

The aim this study focus on understanding the characteristics of specific amino acid residues located in chicken invariant chain (Ii) transmembrane domain in the formation of MHC Ⅱ -Ii complex Two amino acids, Gln47 and Thr50, in transmembrane region of chicken Ii were substituted by Ala via site mutation by the PCR megaprimer method, then the mutated fragment was inserted into pEGFP-C1 vector and the eukaryotic expression vector containing Ii-GFP fusion gene was constructed. At the same time the other eukaryotic expression plasmids, pDsRed2-N1-MHC Ⅱα, pDsRed2-N1-MHC Ⅱ β, pEGFP-N1-MHC Ⅱ α and pEGFP- N1-MHC Ⅱ β were constructed respectively. These recombinant plasmids were solely or co-transfected into COS-7 cells with LipofectamineTM2000. After culture of the cells for 48 h, the expression and intracellular localization of wild type and mutated Ii and MHC Ⅱ subunits were observed with a fluorescent microscope, and their association was analyzed by an immunoprecipitation test. The results showed that the wild type Ii with MHC Ⅱα or MHC Ⅱ β polypeptides could colocalized in cells, while the mutated Ii could not colocalized with MHC Ⅱ α or MHC Ⅱ β in the endocytic compartments. The results of immunoprecipitation showed that the wild type Ii could bind MHC Ⅱ α or (and) MHC Ⅱβ subunits when pEGFP-C1-Ii, pEGFP-N1-MHC Ⅱ α or (and) pEGFP-N1-MHC Ⅱ β were cotransfected in COS-7 cells, while the mutated Ii could not colocalized with MHC Ⅱ α or (and)MHC Ⅱβ subunits when pEGFP-C1-li-Q47A (or pEGFP-C1-Ii-T50A) , pEGFP-N1-MHC Ⅱ α or (and) pEGFP-N1-MHC Ⅱ β were cotransfected in COS-7 cells. Therefore all these results suggest that Gln47 and Thr50 in transmembrane region of chicken Ii play a key role in the assembly of Ii and MHCⅡ subunits.%本研究旨在探明鸡恒定链跨膜区特定氨基酸在形成MHCⅡ-Ii复合物聚合中的作用特征.用大引物PCR定点突变法将鸡Ii链跨膜区2个氨基酸Gln47和Thr50分别突变为丙氨酸(Ala),再连接到pEGFP-C1载体中,构建含Ii-GFP融合基因的真核表达载体.同时还分别构建了含pDsRed2-N1-MHCⅡα、pDsRed2-N1-MHCⅡβ、pEG-FP-N1-MHCⅡa和pEGFP-N1-MHCⅡβ真核表达载体.用脂质体介导法将这些重组质粒单独或共转染至COS-7细胞,培养48 h后经荧光显微镜检测野生型Ii和突变型Ii与MHCⅡ类分子的细胞定位,并通过免疫共沉淀研究它们之间的相互关系.结果显示,野生型Ii链与MHCⅡα或MHCⅡβ之间存在细胞内的共定位,而突变型Ii与MHCⅡα或MHCⅡβ在细胞中共表达后未出现共定位现象.免疫共沉淀结果显示,在野生型Ii链与MHCⅡa或(和)MHCⅡβ单转染或三者共转染COS-7细胞后,均能检测到MHCⅡa-li、MHCⅡβ-Ii和MHCⅡ-Ii复合物,而突变型Ii链与MHCⅡα或(和)MHCⅡβ单转染或三者共转染COS-7细胞后,均检测不到MHCⅡα-Ii、MHCⅡβ-Ii和MHCⅡ-Ii复合物.因此Ii链跨膜区Gln47和Thr50 2个氨基酸对于MHCⅡ-Ii复合体的形成是至关重要的.