人巨细胞病毒M抗原表位的筛选与初步应用研究

发布时间：2018-01-04 04:13

本文关键词：人巨细胞病毒M抗原表位的筛选与初步应用研究　出处：《中国人民解放军军事医学科学院》2007年博士论文　论文类型：学位论文

【摘要】： 人巨细胞病毒属β疱疹病毒亚科，它在人群中感染非常普遍。在成年的健康人群中HCMV的感染呈现不显性临床症状，但对于免疫抑制人群能造成严重疾病，甚至导致生命的危险。目前HCMV临床检测手段并不完善，也没有一种有效的疫苗能够预防病毒的感染。本实验以噬菌体展示技术为手段，以标准羊抗HCMV多抗为靶标，通过正常山羊IgG抗体消减筛选到一个gM抗原表位CMVMME，核心序列为X-XXXX(由于专利问题序列未公开)。经计算机分析及Blast搜索，CMVMME与gM蛋白某一肽段高度同源；Western Blot方法证实鼠抗CMVMME抗体不仅能够特异结合HCMV病毒粒子，也能特异结合重组表达的gM多肽。表明CMVMME是gM的一个线性表位。为初步研究单氨基酸突变对CMVMME结合抗体活性的影响，我们将CMVMME中的关键氨基酸人工单突变为甘氨酸，ELISA和Western Blot结果发现将谷氨酰胺突变为甘氨酸后，CMVMME结合HCMV多抗的活性明显下降，而其它氨基酸单突变后CMVMME活性未见明显变化。提示单氨基酸突变时谷氨酰胺残基影响着CMVMME特异结合HCMV多抗的活性。为证实鼠抗CMVMME多抗具有中和抗体性质，通过病毒中和实验，分别用50μg标准HCMV多抗B65275G、鼠抗CMVMME抗体、正常小鼠抗体和正常山羊抗体处理50TCID_(50)HCMV病毒，发现用鼠抗CMVMME抗体处理后的病毒的感染能力大大降低；用FITC标记病毒即刻早期蛋白表达的方法发现分子量为68KD的病毒即刻早期蛋白在细胞内的表达数量明显减少。标准羊抗HCMV多抗B65275G几乎完全抑制了病毒的侵入，而正常小鼠抗体和正常山羊抗体处理的病毒与不加任何抗体处理的病毒感染能力无明显变化，表明鼠抗CMVMME多抗是一种具有中和作用的抗体，它能在一定程度上阻断HCMV对细胞的侵袭。为了验证CMVMME与HCMV感染的人血清之间的特异性结合反应，首先采用两个不同生物公司生产的HCMV IgG ELISA检测试剂盒分别与40份人血清反应，，发现有8份血清均为HCMV IgG阳性。在这8份IgG阳性人血清中，重组表达的已知的gB抗原表位区AD1能够与5份血清反应，AD2能够与6份血清反应，CMVMME能够与3份血清发生特异性结合反应。将AD1、AD2和CMVMME混合则有7份血清与之反应，表明三种抗原与HCMV抗体反应具有一定的互补性。本研究在HCMV诊断和预防研究上进行了摸索和探讨，取得了一定的结果，这将为进一步研究诊断和预防HCMV的试剂打下良好的基础。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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