本文選題：人巨細(xì)胞病毒　切入點(diǎn)：抗gBn1抗體　出處：《浙江大學(xué)》2008年碩士論文

【摘要】： 研究背景: 人巨細(xì)胞病毒在全世界普遍存在,臨床癥狀主要發(fā)生于免疫系統(tǒng)發(fā)育不全或嚴(yán)重的免疫力缺乏患者。其包膜糖蛋白B(gB)在病毒感染及誘導(dǎo)機(jī)體免疫反應(yīng)中起重要作用,其中g(shù)Bn1型是主要的致病型之一,在我國(guó)的HCMV感染者中占有明顯優(yōu)勢(shì)。 研究目的: 制備能敏感且特異地識(shí)別HCMV gBn1型病毒株的抗體,為今后臨床檢測(cè)HCMV gBn1提供物質(zhì)基礎(chǔ),并為HCMV疫苗的制備及HCMV的基礎(chǔ)研究提供了有力保障。 實(shí)驗(yàn)方法: 根據(jù)GenBank公布的序列(M60929),利用計(jì)算機(jī)輔助設(shè)計(jì),確定并人工合成gBn1抗原多肽,并用Keyhole Limpet Hemocyanin(KLH)交聯(lián)以增強(qiáng)gBn1抗原多肽的免疫原性。將交聯(lián)KLH的gBn1抗原肽免疫新西蘭兔(經(jīng)初次免疫和四次加強(qiáng)免疫)后獲取免疫兔血清,親和純化法純化兔血清中抗gBn1抗體。采用直接ELISA法、免疫細(xì)胞化學(xué)染色法、Western-blot免疫沉淀法鑒定抗gBn1抗體的效價(jià)、反應(yīng)的特異性和敏感性。 實(shí)驗(yàn)結(jié)果: ELISA法、抗原交叉反應(yīng)等實(shí)驗(yàn)顯示,抗gBn1抗體能夠特異地識(shí)別HCMVTowne株(gBn1型),制備的抗體效價(jià)1:64000,且與其它型別的HCMV(AD169株及gBn3株)不發(fā)生反應(yīng)。免疫細(xì)胞化學(xué)染色法鑒定結(jié)果顯示抗gBn1型抗體的特異性與PCR測(cè)定gBn1型基因型的結(jié)果符合,而且與其他兩型(gBn2型和gBn3型)無(wú)交叉反應(yīng)。以HCMV Towne臨床分離株、HCMV AD169病毒株、HCMV gBn3臨床分離株和正常細(xì)胞株為抗原,純化的抗gBn1抗體作為一抗進(jìn)行Western blot分析顯示,抗gBn1抗體與Towne株的制備蛋白反應(yīng)后在相對(duì)分子質(zhì)量約110KD處出現(xiàn)1條清晰條帶,符合HCMV gB的分子量。說(shuō)明所制備的抗體可識(shí)別含有合成多肽相同片斷的HCMV,即gBn1型HCMV,而不識(shí)別其它型別的病毒株,證明抗gBn1抗體具有良好的特異性。 結(jié)論: 本研究制備的抗HCMV gBn1抗體能敏感且特異地識(shí)別HCMV gBn1型病毒株。該抗體在今后HCMV感染的診斷、治療、病毒的結(jié)構(gòu)功能研究以及抗體分析等方面具有良好的應(yīng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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