抗人巨细胞病毒gBn1抗体的制备及鉴定

发布时间：2018-03-31 09:32

  本文选题：人巨细胞病毒　切入点：抗gBn1抗体　出处：《浙江大学》2008年硕士论文

【摘要】： 研究背景: 人巨细胞病毒在全世界普遍存在,临床症状主要发生于免疫系统发育不全或严重的免疫力缺乏患者。其包膜糖蛋白B(gB)在病毒感染及诱导机体免疫反应中起重要作用,其中gBn1型是主要的致病型之一,在我国的HCMV感染者中占有明显优势。 研究目的: 制备能敏感且特异地识别HCMV gBn1型病毒株的抗体,为今后临床检测HCMV gBn1提供物质基础,并为HCMV疫苗的制备及HCMV的基础研究提供了有力保障。 实验方法: 根据GenBank公布的序列(M60929),利用计算机辅助设计,确定并人工合成gBn1抗原多肽,并用Keyhole Limpet Hemocyanin(KLH)交联以增强gBn1抗原多肽的免疫原性。将交联KLH的gBn1抗原肽免疫新西兰兔(经初次免疫和四次加强免疫)后获取免疫兔血清,亲和纯化法纯化兔血清中抗gBn1抗体。采用直接ELISA法、免疫细胞化学染色法、Western-blot免疫沉淀法鉴定抗gBn1抗体的效价、反应的特异性和敏感性。 实验结果: ELISA法、抗原交叉反应等实验显示,抗gBn1抗体能够特异地识别HCMVTowne株(gBn1型),制备的抗体效价1:64000,且与其它型别的HCMV(AD169株及gBn3株)不发生反应。免疫细胞化学染色法鉴定结果显示抗gBn1型抗体的特异性与PCR测定gBn1型基因型的结果符合,而且与其他两型(gBn2型和gBn3型)无交叉反应。以HCMV Towne临床分离株、HCMV AD169病毒株、HCMV gBn3临床分离株和正常细胞株为抗原,纯化的抗gBn1抗体作为一抗进行Western blot分析显示,抗gBn1抗体与Towne株的制备蛋白反应后在相对分子质量约110KD处出现1条清晰条带,符合HCMV gB的分子量。说明所制备的抗体可识别含有合成多肽相同片断的HCMV,即gBn1型HCMV,而不识别其它型别的病毒株,证明抗gBn1抗体具有良好的特异性。 结论: 本研究制备的抗HCMV gBn1抗体能敏感且特异地识别HCMV gBn1型病毒株。该抗体在今后HCMV感染的诊断、治疗、病毒的结构功能研究以及抗体分析等方面具有良好的应用前景。
[Abstract]:Background:. Human cytomegalovirus (HCMV) is prevalent all over the world. Its clinical symptoms mainly occur in patients with hypoplasia of the immune system or severe immune deficiency. Its envelope glycoprotein (BGB) plays an important role in viral infection and inducing immune response. Among them, gBn1 type is one of the main pathogenic forms, and has obvious advantage in HCMV infection in our country. Objectives of the study:. The preparation of antibodies that can recognize HCMV gBn1 virus strains sensitively and specifically provides a material basis for the future clinical detection of HCMV gBn1, and provides a strong guarantee for the preparation of HCMV vaccine and the basic research of HCMV. Experimental methods:. According to the sequence M60929 published by GenBank, the peptide of gBn1 antigen was determined and synthesized by computer aided design. The immunogenicity of gBn1 antigenic peptides was enhanced by Keyhole Limpet hemocyanin (KLH) crosslinking. The immunized New Zealand rabbits were immunized with cross-linked KLH gBn1 antigenic peptides (after primary and four times enhanced immunization). The anti gBn1 antibody in rabbit serum was purified by affinity purification, and the titer, specificity and sensitivity of anti gBn1 antibody were identified by direct ELISA method and immunocytochemical staining and Western-blot immunoprecipitation. Experimental results:. ELISA assay, antigenic cross reaction and other experiments showed that, Anti gBn1 antibody can specifically recognize HCMVTowne strain gBn1, the titer of the prepared antibody is 1: 64000, and it does not react with other types of HCMV(AD169 and gBn3 strains. Immunocytochemical staining showed that the specificity of anti gBn1 antibody was compared with that of PCR. The results of genotyping of gBn1 were consistent. There was no cross reaction with the other two types of gBn3 and gBn2). The clinical isolates of HCMV Towne and normal cell lines were used as antigens, and the purified anti-#en5# antibodies were analyzed by Western blot analysis. There was a clear band at the relative molecular weight of 110KD after the reaction of the anti- gBn1 antibody with the prepared protein of the Towne strain. In accordance with the molecular weight of HCMV GB, the prepared antibody can recognize HCMV containing the same fragment of synthetic polypeptide, that is, gBn1 type, but not other types of virus strains. It is proved that the anti-HCV antibody has good specificity. Conclusion:. The anti HCMV gBn1 antibody prepared in this study can recognize HCMV gBn1 virus strain sensitively and specifically. The antibody has a good application prospect in the diagnosis, treatment, structure and function study and antibody analysis of HCMV infection in the future.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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