中文摘要：

恒定链（invariant chain,Ii）能结合主要组织相容性复合体（major histocompatibility complex,MHC）Ⅰ类和Ⅱ类分子,并在MHC递呈抗原中起重要辅助作用。为了探讨鱼类Ii和MHC分子体外结合关系,本研究采用Pull-down方法检测草鱼（Ctenopharyngodon idellus）Ii与MHCⅠ类或Ⅱ类分子的结合。克隆草鱼Ii（711 bp）、MhcⅠα（540 bp）和MhcⅡβ（549 bp）基因片段,再将其分别插入原核表达质粒PET-22b（无His标签）或PET-28a（含His标签）中,构建了3个重组质粒PET-22b-Ii、PET-28a-MhcⅠα和PET-28aMhcⅡβ。然后分别转染工程菌大肠杆菌（Escherichia coli）Rosetta（DE3）,进行诱导表达、纯化和聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis,PAGE）鉴定。结果表明,含His标签的MHCⅠα和MHCⅡβ不仅表达,而且可高效纯化,其相对分子质量约为24 k D;Western blot结果表明,Ii可表达,相对分子质量约为30 k D。将PET-22b-Ii与PET-28a-MhcⅠα或PET-28a-MhcⅡβ分别共转染Rosetta（DE3）,构建了重组菌Rosetta（DE3）（PET-28a-MhcⅠα＋PET-22b-Ii）和Rosetta（DE3）（PET-28a-MhcⅡβ＋PET-22b-Ii）。经诱导表达和PAGE检测发现,MHCⅠα和MHCⅡβ分别结合Ii,形成相对分子质量为54 k D的复合物;SDS处理后,分别被解离形成单分子Ii（30 k D）以及MHCⅠα或MHCⅡβ（24 k D）。在Western blot中,解离后的Ii可与特异性多克隆抗体结合并被电化学发光（electrogenerated chemiluminescence,ECL）显色。综上所述,草鱼Ii可以分别与MHCⅡβ和MHCⅠα结合,为进一步研究动物Ii与MHC分子的关系提供了基础资料。

英文摘要：

Invariant chain （Ii） can bind to major histocompatibility complex （MHC） class Ⅰ or Ⅱ molecules, and plays an important assistant role in antigen presentation by MHC molecules. In order to explore what about the binding of Ii with MHC molecules, a Pull-down method for detection of association of grass carp （Ctenopharyngodon idellus） Ii with MHC molecules was reported in the present study. DNA segments of Ii （711 bp）, Mhc Ⅰα （540 bp） and Mhc Ⅱβ （549 bp） in grass carp were cloned, and then respectively inserted into prokaryotic expression plasmid PET-22b （no His-tag） or PET-28a （carry with His-tag） to construct 3 recombinant plasmids of PET-22b-Ii, PET-28a-Mhc Ⅰα and PET-28a-Mhc Ⅱβ. The above 3 recombinant plasmids then were transfected into engineering bacteria （Escherichia coli） Rosetta （DE3）, respectively. After an induction to express and renature the interest proteins and purification by Ni ＋-NTA affinity chromatography column, the purified expressed protein products were detected by polyacrylamide gel electrophoresis （PAGE）. The results indicated that MHC Ⅰα and MHCⅡβ labeled with His-tag could well express and be purified each with a molecular weight of 24 kD. The result of Western blot showed that Ii also well expressed and had a molecular weight of about 30 kD. Finally, the recombinant plasmid PET-22b-Ii was cotransfected with PET-28a-Mhc Ⅰα or PET-28a-Mhc Ⅱβ into Rosetta （DE3）, respectively, to construct 2 recombinant strains of Rosetta（DE3 ）（PET- 28a-MhcⅠα ＋ PET- 22b-Ii） and Rosetta（DE3 ）（PET- 28a-Mhc Ⅱβ＋ PET-22b-Ii）. Induced expression and PAGE detection showed that MHCⅠα or MHC Ⅱβ could bind Ii to form a complex of 54 kD, respectively, which could be dissociated into simple molecule of Ii （30 kD）, MHC Ⅰα or MHC Ⅱβ （24 kD） after SDS treatment, respectively. Western blot result showed that the dissociated Ii could bind specific antibody and be coloured by electrogenerated chemilumine