本文選題：C5a　切入點(diǎn)：CD88　出處：《第三軍醫(yī)大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 過敏毒素C5a是補(bǔ)體系統(tǒng)活化產(chǎn)物,也可由一些吞噬性細(xì)胞產(chǎn)生,它是炎癥反應(yīng)的重要介質(zhì)和趨化因子,在急性肺損傷、膿毒血癥、腦膜炎、風(fēng)濕性關(guān)節(jié)炎、腎小球腎炎等多種疾病的發(fā)生發(fā)展過程中起重要作用。因此,抑制C5a的生物學(xué)活性對(duì)于以上炎性疾病的治療與預(yù)防有著重要的意義。 目前的C5a抑制劑大多以C5a受體(CD88)為靶位,通過與C5a競(jìng)爭(zhēng)性結(jié)合CD88而達(dá)到抑制C5a生物活性的目的。本研究關(guān)注C5a受體上同C5a結(jié)合的關(guān)鍵位點(diǎn),從中篩選既能直接和配體C5a結(jié)合,又能拮抗配體C5a生物活性的新型C5a抑制劑,并在此基礎(chǔ)上,對(duì)其功能作了初步研究。本研究由3個(gè)部分組成: 第一部分:C5a結(jié)合肽的篩選 利用噬菌體展示肽庫(kù)技術(shù),首先從噬菌體展示十二肽庫(kù)篩選陽(yáng)性克隆,以rhC5a為包被靶分子,將噬菌體展示十二肽庫(kù)與包被有靶分子的平板共溫育,先洗去未結(jié)合的噬菌體,然后洗脫與rhC5a特異性結(jié)合的噬菌體,將洗脫下來的rhC5a特異性結(jié)合的噬菌體進(jìn)行擴(kuò)增,再進(jìn)行下一輪的結(jié)合/擴(kuò)增循環(huán),以富集那些可結(jié)合序列。 3次重復(fù)以上過程,測(cè)序所得陽(yáng)性克隆,從而得到其氨基酸序列。我們從噬菌體展示十二肽庫(kù)得到的陽(yáng)性克隆中隨機(jī)挑取151克隆進(jìn)行測(cè)序鑒定。 為了更精確地確認(rèn)C5a與其受體的結(jié)合位點(diǎn),期望找到更精細(xì)的拮抗多肽,同時(shí)考慮環(huán)化肽結(jié)構(gòu)的穩(wěn)定性,針對(duì)噬菌體展示十二肽庫(kù)的實(shí)驗(yàn)結(jié)果,本研究又以上述相同的方法,從受限于二硫鍵環(huán)內(nèi)的噬菌體展示七肽庫(kù)篩選陽(yáng)性克隆,同樣以rhC5a為包被靶分子,從受限于二硫鍵環(huán)內(nèi)的噬菌體展示七肽庫(kù)中我們共挑取138陽(yáng)性克隆并測(cè)序鑒定。 第二部分:C5a受體衍生肽的確定 應(yīng)用軟件Vector NTI Suite 9對(duì)以上得到的多肽序列同C5a受體(分別是CD88、C5L2)氨基酸序列進(jìn)行比對(duì),分析其與C5a受體匹配區(qū)域的同源性,從高同源性序列中得到的一系列多肽,我們定義為C5a受體衍生肽。 運(yùn)用EXPASY工具,分析C5L2的二級(jí)結(jié)構(gòu),結(jié)合陽(yáng)性克隆比對(duì)結(jié)果,我們分析了
[Abstract]:The allergy toxin C5a is an active product of the complement system and can also be produced by some phagocytic cells. It is an important mediator and chemokine in inflammatory response in acute lung injury, sepsis, meningitis, rheumatoid arthritis, The inhibition of biological activity of C5a plays an important role in the occurrence and development of many diseases such as glomerulonephritis. Therefore, the inhibition of biological activity of C5a plays an important role in the treatment and prevention of the above inflammatory diseases. Most of the current C5a inhibitors target C5a receptor CD88, which can inhibit the biological activity of C5a by competitive binding with C5a. This study focused on the key sites of C5a receptor binding to C5a, and screened the C5a binding directly to the ligand C5a. A novel C5a inhibitor, which antagonizes the biological activity of ligand C5a, has been studied on the basis of which the function of C5a inhibitor has been preliminarily studied. Part one: screening of C5a binding peptides. The phage display peptide library was used to screen the positive clones from the phage display 12 peptide library. The phage display 12 peptide library was incubated with the plate coated with the target molecule, and the unbound phage was first washed out. Then we elucidate the rhC5a specific phage, then amplify the eluted rhC5a specific phage, and then carry on the next cycle of binding / amplification to enrich those binding sequences. The positive clones were sequenced three times and the amino acid sequence was obtained. We randomly selected 151 clones from the phage display 12 peptide library for sequencing. In order to identify the binding site of C5a to its receptor more accurately, and to find more precise antagonistic peptides, and to consider the stability of cyclic peptide structure, we used the same method to display the 12 peptide library by phage display. The positive clones were screened from the phage display heptapeptide library limited by disulfide bond ring, and the rhC5a was also used as the encapsulated target molecule. We selected 138 positive clones from the phage display heptapeptide library limited by disulfide bond ring and identified them by sequencing. Part two: determination of the receptor derivative peptide of: C5a. Vector NTI Suite 9 was used to compare the above peptide sequences with the amino acid sequence of C5a receptor (CD88A C5L2), and to analyze the homology of the matching region with C5a receptor, and a series of polypeptides obtained from high homology sequence. We define C5a receptor-derived peptides. Using EXPASY tool, the secondary structure of C5L2 was analyzed. Combined with the results of positive clone comparison, we analyzed the structure of C5L2.
【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
【分類號(hào)】：R341

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