本文關(guān)鍵詞： cubilin 同源模建 原核表達(dá) 親和層析　出處：《第三軍醫(yī)大學(xué)》2006年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 腎小管上皮細(xì)胞超負(fù)荷重吸收白蛋白可通過(guò)多種途經(jīng)損傷腎組織,最終導(dǎo)致慢性腎功能衰竭[1,2]。Cubilin是分布于腎近曲小管上皮細(xì)胞刷狀緣負(fù)責(zé)重吸收白蛋白等大分子物質(zhì)的吞飲受體[3-5]。在cubilin缺陷的人和狗,均出現(xiàn)大量白蛋白尿,但腎組織無(wú)明顯病理改變[6-8],也不會(huì)進(jìn)展為慢性腎功能衰竭[9]。我們的研究表明,腎病綜合征患者腎小管上皮細(xì)胞cubilin表達(dá)上調(diào),重吸收白蛋白增加,與腎小管間質(zhì)MCP-1和RANTES表達(dá)增加和炎癥細(xì)胞浸潤(rùn)的改變相關(guān)[10],而反義cubilin RNA,在抑制白蛋白超負(fù)荷重吸收的同時(shí),也明顯抑制腎小管上皮細(xì)胞MCP-1和RANTES的表達(dá)[11],提示cublilin在介導(dǎo)白蛋白超負(fù)荷引起的腎小管間質(zhì)損害中具有重要作用。Cubilin全長(zhǎng)3623aa,460kD,由N-端,8個(gè)EGF樣結(jié)構(gòu)和27個(gè)CUB結(jié)構(gòu)域組成[12],CUB結(jié)構(gòu)域是主要的功能域,負(fù)責(zé)和絕大部分的配體結(jié)合,其中CUB7-8結(jié)構(gòu)域是白蛋白的結(jié)合域,但二者結(jié)合的關(guān)鍵氨基酸殘基尚未確定[13]。由于蛋白質(zhì)之間的相互作用是通過(guò)分子內(nèi)部的肽段形成特殊空間結(jié)構(gòu)實(shí)現(xiàn)的(如“配體結(jié)合口袋”負(fù)責(zé)受體與配體間的識(shí)別與結(jié)合),通常只需幾個(gè)關(guān)鍵功能殘基決定其相互作用的效率。針對(duì)Cubilin各段生物學(xué)功能不同的特性,選取能夠與白蛋白結(jié)合的CUB7-8功能片段為研究對(duì)象,以同源模建方法建立CUB8功能域的三維模型,并以計(jì)算機(jī)輔助的分子對(duì)接方法預(yù)測(cè)CUB8功能域與白蛋白的候選結(jié)合氨基酸殘基,通過(guò)原核表達(dá)野生型和突變型CUB7-8蛋白片段,利用生物傳感器技術(shù),測(cè)定野生型和突變型CUB7-8蛋白片段與白蛋白的親和力,以確定cubilin與白蛋白結(jié)合的關(guān)鍵氨基酸殘基,為進(jìn)一步研究cubilin的生理功能和病理生理意義奠定基礎(chǔ)。實(shí)驗(yàn)方法 1.利用NCBI和PDB數(shù)據(jù)庫(kù),搜索CUB8的氨基酸序列及與該片段相似性較高且已經(jīng)解析三維結(jié)構(gòu)的模板蛋白,再利用insight II工作站對(duì)CUB8蛋白片段進(jìn)行同源模建,然后對(duì)得到的CUB8蛋白片段和白蛋白進(jìn)行分子對(duì)接,尋找白蛋白和CUB8的候選結(jié)合氨基酸殘基。
[Abstract]:Renal tubule epithelial cells overload and reabsorption of albumin can lead to chronic renal failure through multiple ways to damage renal tissue. [Cubilin is a receptor that reabsorbs albumin and other macromolecules in the brush border of renal proximal tubule epithelial cells. [3-5] .A large amount of albuminuria was found in both human and dog with cubilin deficiency, but there were no obvious pathological changes in renal tissue. [6-8], and it doesn't progress to chronic renal failure. [9. Our study showed that the expression of cubilin in renal tubular epithelial cells was up-regulated and the reabsorption of albumin was increased in patients with nephrotic syndrome. Correlated with increased expression of MCP-1 and RANTES in renal tubulointerstitium and changes of inflammatory cell infiltration. [While antisense cubilin RNAs inhibited albumin overload and reabsorption, it also inhibited the expression of MCP-1 and RANTES in renal tubular epithelial cells. [It is suggested that cublilin plays an important role in mediating tubulointerstitial damage induced by albumin overload. Composition of 8 EGF like structures and 27 CUB domains. [12 the cub domain is the main domain responsible for binding to most of the ligands, in which the CUB7-8 domain is the binding domain of albumin, but the key amino acid residues of the two binding domains have not been determined. [13. Since the interaction between proteins is achieved through the formation of special spatial structures of peptide segments within molecules (e.g. "ligand binding pockets", which are responsible for the recognition and binding between receptors and ligands). Usually only a few key functional residues are required to determine the efficiency of their interactions. The functional fragments of CUB7-8 which can bind to albumin were selected as the research object, and the 3D model of CUB8 functional domain was established by homologous modeling method. The candidate amino acid residues of CUB8 domain and albumin were predicted by computer assisted molecular docking method, and the wild-type and mutants CUB7-8 protein fragments were expressed by prokaryotic expression. The affinity of wild-type and mutant CUB7-8 protein fragments to albumin was determined by biosensor technique to determine the key amino acid residues of cubilin binding to albumin. To lay a foundation for further study of physiological function and pathophysiological significance of cubilin. 1. NCBI and PDB databases were used to search for the amino acid sequence of CUB8 and the template protein with high similarity to the fragment. Insight II workstation was used for homologous modeling of CUB8 protein fragment, and then the CUB8 protein fragment and albumin were docked. To search for candidate binding amino acid residues of albumin and CUB8.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R341

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