中文摘要：

本研究旨在探明鸡（Gallus gallus）主要组织相容性复合体（major histocompatibility complex,MHC）Ⅱ类分子（B分子）中与恒定链（invariant chain,Ii）结合的活性片段,以了解和深入研究Ⅱ类分子与Ii在递呈抗原肽中的作用机理。从实验室保存的B-LB基因的c DNA克隆了3个结构片段分别插入原核和真核表达质粒,再将其分别转染或者与Ii共转染工程菌Rosetta（DE3）,诱导表达后先后用亲和层析和拉下法（pull-down）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE）检测其纯度和形成的复合物。然后用免疫印迹（Western blot）验证复合物的性质以及真核表达验证这些片段在细胞内的表达和定位。首先,构建的3个重组质粒p GEX-4T-1-B-LB-Sβ1、p GEX-4T-1-B-LB-β1和p GEX-4T-1-B-LB-β2TC均能单一表达目的蛋白B-LB-Sβ1、B-LB-β1和B-LB-β2TC,并被纯化。其次,在共表达和pull-down中,B-LB-Sβ1和B-LB-β1能够与Ii形成复合物,而B-LB-β2TC不能与Ii结合,因为免疫印迹的结果表明,特异性抗体能识别在poll-down中被结合的这两个目的蛋白。最后在真核表达系统中,也证实B-LB-Sβ1、B-LB-β1能保持B-L分子在293T细胞的浆膜系统定位的功能,而B-LB-β2TC却丧失了该定位功能。本研究结果首次提供了Ⅱ类分子结合Ii的活性片段的证据。

英文摘要：

This work focus on clearing active segments of chicken（Gallus gallus） major histocompatibility complex（MHC） class Ⅱ molecules binding with invariant chain（Ii） for research of mechanism of MHC and Ii action in presenting antigen peptides. To this end 3 DNA segments（Sβ1, β1 and β2TC） were cloned from c DNA of B-LB gene, which was kept in our laboratory, and inserted into prokaryotic or eukaryotic expression plasmids respectively. Then these recombinant plasmids were respectively transfected or co-transfected with Ii into engineering bacteria, Rosetta（DE3）. The expression products or complexes（B-LB segment and Ii） were purified by affinity chromatography and identified by pull-down and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS- PAGE）. Moreover, the expression and localization of B- LB segments and Ii in the eukaryotic cells were observed too. The results showed that first the 3 recombinant plasmids（p GEX-4T-1-BLB-Sβ1, p GEX-4T-1-B-LB-β1 and p GEX-4T-1-B-LB-β2TC） could well express, and the interest proteins（B-LB- Sβ1, B- LB- β1 and B- LB- β2TC） also well be purified by an affinity chromatography. Secondly the segment B-LB-Sβ1 or B-LB-β1 rather than B-LB-β2TC bound His/Ii, which adsorbed to Ni-column, forming complexes by the co-transfection and followed pull-down, because in the Western blot the 3 interest proteins could be recognized by specific antibody in the co- transfected products, but only B- LB- Sβ1 and B- LB- β1were found as the complexes（His/Ii and B-LB-Sβ1, His/Ii and B-LB-β1）, which were dissociated from Nicolumn in pull- down. Finally it was proved that B- LB- Sβ1 and B- LB- β1 could express and localize in the endometrial system of 293 T cells, which kept the function such as whole MHC molecule, but the B-LB-β2TC lost this function. All of above results first time provide an evidence of binding of active segments of MHC class Ⅱmolecules with Ii in vitro.