CR3的Ⅰ结构域的克隆和在CHO细胞中的表达及配体结合特性的研究

STUDIES ON Ⅰ DOMAIN OF CD11b: CLONING OF THE cDNA, EXPRESSION IN CHO CELLS, AND ITS ROLE IN LIGAND BINDING

目的研究白细胞整合素ＣＲ３α亚基的Ⅰ结构域在配体结合中的作用。方法克隆ＣＤ１１ｂ的Ⅰ结构域，用重叠ＰＣＲ的方法将其与信号肽ＤＮＡ连接，克隆到表达载体ＰＥＥ６ＨＣＭＶ．ＧＳ，转化ＣＨＯ－Ｋ１细胞。表达的Ⅰ－结构域经ＳＰ－Ｓｅｐｈａｒｏｓｅ和ｍｏｎｏ－Ｓ柱纯化后，与Ｃ３ｂｉ进行配体结合实验。结果用ＳＤＳ－ＰＡＧＥ测得重组的Ⅰ结构域分子量为２９ｋＤ。重组的Ⅰ结构域可以与Ｃ３ｂｉ结合，Ｓｃａｔｃｈａｒｄ分析示结合常数为３００±１１３ｎｍｏｌ／Ｌ，这种结合可以被抗Ⅰ－结构域单克隆抗体ＬＰＭ１９Ｃ和ＥＤＴＡ抑制。结论Ⅰ结构域对于结合单体Ｃ３ｂｉ是必要的和充分的，结合需要二价阳离子。

Objective To investigate the role of Ⅰ-domain in the ligand binding of CR3(CD11b). Methods The cDNA of the Ⅰ domain and the leader sequence of CD11b were joined together by overlap extension. The construct was cloned into pEE6HCMV.GS vector and transformed into CHO-K1 cells by calcium phosphate mediate precipitation. The recombinant Ⅰ domain was purified on SP-sepharose and mono-S columns. The purified Ⅰ domain was coated on Terasaki plate for measuring the binding of C3bi-AP to the protein.Results SDS-PAGE analysis of recombinant Ⅰ domain showed a major band at 29 kD. The binding affinity of C3bi-AP to Ⅰ domain was 300±113nmol/L as estimated by Scatchard analysis. The Ⅰ domain dependent binding was inhibited by mAb LPM19C (anti-Ⅰ domain ) and EDTA. Conclusion The Ⅰ domain is necessary and sufficient for binding monomeric C3bi and the binding is divalent cation-dependent