IgA1蛋白的體外組裝及其在誘導人細胞分泌素產生的研究

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题名:	IgA1蛋白的體外組裝及其在誘導人細胞分泌素產生的研究
其它题名:	Studies on Molecular Assembly of IgA1 Protease and Its Role in Production of Cytokines
作者:	Shiping He(何世屏);林雅玲(Lin,Ya-Ling)
贡献者:	醫學院醫學系學士班寄生蟲學科
关键词:	IgA1 蛋白;分子體外組裝;細胞分泌素;定點突變;分子識別;IgA1 protease;in vitro molecular assembly;Cytokines;Site-directed mutagenesis;Molecular recognition
日期:	2003-07-31
上传时间:	2009-09-01 16:18:11 (UTC+8)
摘要:	大量的研究證明﹕包括流行性感冒噬血性桿菌在內的致病性細菌所產生的 IgA1 蛋白對於細菌的感染性及菌落形成之重要性與其的含量成正比。此一方面可以直接導致人體第一線防衛系統的衰減﹐另一方面又可誘導包括 IL-1TNF-αIL-6 和 IL-8 等在內的細胞分泌素的形成﹐對於細菌的感染在感染區迅速形成菌落及最終導致內部發炎等起著極為重要的作用。在以往的研究中﹐我們已從 non-typable 流行性感冒噬血性桿菌中克隆了 iga 基因﹐並分別表達及純化了 IgA1 蛋白的α-及β-鏈﹐還在體外實驗中證明瞭重組的α-鏈須在β-鏈存在時才能具有更好的蛋白之活性。本研在此基礎上進一步研究了 IgA1 蛋白中二條鏈的組裝方式的分子機制。為了研究分子識別, 我們完成了分子內二條鏈(α-鏈及β-鏈)中八個抗體識別殘基中的六個的突變體, 為第 93 年度的研究(即完成剩餘的二個突變體的分子識別及對細胞分泌素的誘導方式等)打下了基礎。突變體基因制備完成後已克隆到表達載體上並獲得了突變基因的表現。純化的突變體及正常所組裝雜合分子對人體 IgA1 水解的結果表明, 抗體分子識別位點的殘基的確對分子組裝及其對人體免疫球蛋白水解活性均有明顯的影響。 IgA1 protease is an important protease produced by pathogenic bacteria including Haemophilus influenzae, the pathogenic potency of which is related to the relative amounts of the protease. This protease has also been found to induce cytokine production, such as IL-1, TNF- , IL-6 and IL-8, suggesting that IgA1 protease is involved in both neutralising host defence molecule, IgA1, and the production of cytokines which contributes to infection of bacteria and inflammation at the sites of infection. In our previous study, we have achieved cloning, expression and purification of the recombinant .alpha.-chain (r.alphia.-chain), the surface domain of .beta.-chain (sr.beta.-chain) and the whole domain of .beta.-chain (r.beta.-chain). Interaction between the r.alpha.-chain and the sr.beta.-chain showed that the assembly of the two chains enhanced the proteolytic activity of the r.alpha.-chain. Also, we have gained antibodies to both .alpha.- and .beta.-chain. In order to study how and why the .beta.-chain of the protease enhances proteolytic activities of the .alpha.-chain and the role of IgA1 protease in infection and inflammation, with the aid of an NSC grant (NSC92-2311-B-110-006), we have created 6 of the 8 estimated residues from the antibody binding sites on both domains. Mutant DNA has been expressed with six-histidine tag in E. coli, a non-IgA1 protease production host cell, and the mutant enzyme has been purified to homology with affinity chromatography. In vitro assembly and proteolytic assays showed that the mutants are indeed affected the molecular assembly of the protease and thus the proteolytic activity ranging from 10-1,000-fold.
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