本文關(guān)鍵詞：人巨細(xì)胞病毒M抗原表位的篩選與初步應(yīng)用研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2007年博士論文　論文類型：學(xué)位論文

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【摘要】： 人巨細(xì)胞病毒屬β皰疹病毒亞科，它在人群中感染非常普遍。在成年的健康人群中HCMV的感染呈現(xiàn)不顯性臨床癥狀，但對于免疫抑制人群能造成嚴(yán)重疾病，甚至導(dǎo)致生命的危險。目前HCMV臨床檢測手段并不完善，也沒有一種有效的疫苗能夠預(yù)防病毒的感染。 本實驗以噬菌體展示技術(shù)為手段，以標(biāo)準(zhǔn)羊抗HCMV多抗為靶標(biāo)，通過正常山羊IgG抗體消減篩選到一個gM抗原表位CMVMME，核心序列為X-XXXX(由于專利問題序列未公開)。經(jīng)計算機分析及Blast搜索，CMVMME與gM蛋白某一肽段高度同源；Western Blot方法證實鼠抗CMVMME抗體不僅能夠特異結(jié)合HCMV病毒粒子，也能特異結(jié)合重組表達(dá)的gM多肽。表明CMVMME是gM的一個線性表位。 為初步研究單氨基酸突變對CMVMME結(jié)合抗體活性的影響，我們將CMVMME中的關(guān)鍵氨基酸人工單突變?yōu)楦拾彼幔珽LISA和Western Blot結(jié)果發(fā)現(xiàn)將谷氨酰胺突變?yōu)楦拾彼岷�，CMVMME結(jié)合HCMV多抗的活性明顯下降，而其它氨基酸單突變后CMVMME活性未見明顯變化。提示單氨基酸突變時谷氨酰胺殘基影響著CMVMME特異結(jié)合HCMV多抗的活性。 為證實鼠抗CMVMME多抗具有中和抗體性質(zhì)，通過病毒中和實驗，分別用50μg標(biāo)準(zhǔn)HCMV多抗B65275G、鼠抗CMVMME抗體、正常小鼠抗體和正常山羊抗體處理50TCID_(50)HCMV病毒，發(fā)現(xiàn)用鼠抗CMVMME抗體處理后的病毒的感染能力大大降低；用FITC標(biāo)記病毒即刻早期蛋白表達(dá)的方法發(fā)現(xiàn)分子量為68KD的病毒即刻早期蛋白在細(xì)胞內(nèi)的表達(dá)數(shù)量明顯減少。標(biāo)準(zhǔn)羊抗HCMV多抗B65275G幾乎完全抑制了病毒的侵入，而正常小鼠抗體和正常山羊抗體處理的病毒與不加任何抗體處理的病毒感染能力無明顯變化，表明鼠抗CMVMME多抗是一種具有中和作用的抗體，它能在一定程度上阻斷HCMV對細(xì)胞的侵襲。 為了驗證CMVMME與HCMV感染的人血清之間的特異性結(jié)合反應(yīng)，首先采用兩個不同生物公司生產(chǎn)的HCMV IgG ELISA檢測試劑盒分別與40份人血清反應(yīng)，，發(fā)現(xiàn)有8份血清均為HCMV IgG陽性。在這8份IgG陽性人血清中，重組表達(dá)的已知的gB抗原表位區(qū)AD1能夠與5份血清反應(yīng)，AD2能夠與6份血清反應(yīng)，CMVMME能夠與3份血清發(fā)生特異性結(jié)合反應(yīng)。將AD1、AD2和CMVMME混合則有7份血清與之反應(yīng)，表明三種抗原與HCMV抗體反應(yīng)具有一定的互補性。 本研究在HCMV診斷和預(yù)防研究上進(jìn)行了摸索和探討，取得了一定的結(jié)果，這將為進(jìn)一步研究診斷和預(yù)防HCMV的試劑打下良好的基礎(chǔ)。
[Abstract]:Human cytomegalovirus belongs to 尾 herpesvirus subfamily, it is very common infection in the population. In adult healthy people, HCMV infection shows non-dominant clinical symptoms, but it can cause serious disease in immunosuppressive population. At present, the clinical test of HCMV is not perfect, and there is no effective vaccine to prevent virus infection. In this experiment, a GM epitope CMVMME was screened by using phage display technique and standard goat anti HCMV polyclonal antibody as target, by subtractive antibody to normal goat IgG. The core sequence was X-XXXX.Compared with the patent problem, the sequence was highly homologous to a peptide of GM protein by computer analysis and Blast search. Western Blot method confirmed that mouse anti CMVMME antibody could not only specifically bind to HCMV virus particles. CMVMME is a linear epitope of GM. In order to study the effect of single amino acid mutation on the activity of CMVMME binding antibody, we transformed the key amino acid in CMVMME into glycine. The results of ELISA and Western Blot showed that the activity of MME binding to HCMV was significantly decreased after glutamine was mutated to glycine. However, the activity of CMVMME did not change significantly after single amino acid mutation, suggesting that glutamine residues affect the activity of CMVMME specific binding HCMV polyclonal antibodies. In order to confirm the neutralizing antibody properties of mouse anti CMVMME polyantibody, 50 渭 g standard HCMV polyclonal antibody B65275G and mouse anti CMVMME antibody were used in virus neutralization test. Normal mouse antibody and normal goat antibody were used to treat 50TCIDD virus. It was found that the infection ability of the virus treated with mouse anti CMVMME antibody was greatly reduced. The expression of immediate early protein of virus with molecular weight of 68KD was significantly reduced by FITC labeling method. Standard Sheep anti HCMV polyclonal antibody B65275G. Almost completely inhibited the virus's invasion. However, the infection ability of the virus treated with normal mouse antibody and goat antibody was not significantly different from that of the virus without any antibody treatment, which indicated that the mouse anti CMVMME polyantibody was a neutralizing antibody. It can block the invasion of HCMV to some extent. In order to verify the specific binding reaction between CMVMME and human serum infected with HCMV. First, HCMV IgG ELISA kit produced by two different biological companies was used to react with 40 human serum samples. Eight sera were found to be HCMV IgG positive, and 5 of the 8 IgG positive sera were able to react with five known GB epitope regions expressed by recombinant AD1. AD2 could react with 6 serum samples and 3 sera, and 7 sera with AD1 + AD2 and CMVMME. The results showed that the three antigens were complementary to HCMV antibodies. In this study, the diagnosis and prevention of HCMV have been explored and discussed, and some results have been obtained, which will lay a good foundation for the further study of the reagent for diagnosis and prevention of HCMV.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王本旭;劉展;劉玉;沈倍奮;柳川;邵寧生;;人巨細(xì)胞病毒M抗原表位保守氨基酸突變的分析[J];生物工程學(xué)報;2008年07期

本文編號：1376974