甲肝病毒L-8株单克隆抗体的制备与鉴定

发布时间：2018-04-29 15:51

本文选题：甲肝病毒 + 杂交瘤细胞株　；参考：《昆明医学院》2006年硕士论文

【摘要】：甲型肝炎是危害人类健康的传染病，其病原体为甲型肝炎病毒(Hepatitis A virus；HAV)，属于小RNA病毒科毒粒，只有一个血清型和多个基因亚型。甲型肝炎在发展中国家多年来都有不同程度的流行，甲肝减毒活疫苗和灭活疫苗的使用，为甲肝的预防提供了可靠的手段，但偶有在接种疫苗后发生甲型肝炎的案例，这就需要对患者所感染的病原体进行鉴别，以明确患者发病的原因是接种疫苗引发还是甲肝野毒株感染导致的偶合病例。1975年KOhler和Milstein建立了杂交瘤技术制备了单克隆抗体(monoclonal antibody；McAb)。单克隆抗体是针对单一抗原决定簇的均一的特异的抗体，具有高度的特异性，被应用于病毒不同型或同型不同株间的抗原关系分析，具有特异性强、灵敏度高，成本低，重复性好等优点，因此，我们可以采用单克隆抗体对甲肝病毒的不同株进行鉴别。甲肝病毒L-8株是我所在1988年上海甲肝暴发流行时分离出来的毒株，属于IB亚型，目前已制备出灭活疫苗并在全国广泛使用。我们选择与我所产品相关的甲肝病毒L-8株作为免疫原，免疫BALB／C小鼠，取免疫后的脾细胞与骨髓瘤细胞(SP2／0)进行融合，通过HAT选择性培养系统培养，ELISA法检测抗体筛选阳性克隆，并对阳性克隆进行有限稀释法克隆化培养，最后筛选出一株稳定分泌抗HAV特异性抗体的杂交瘤细胞株，其上清和腹水效价分别为1：64；1：10000；杂交细胞染色体计数为80～90，，证明是两种细胞杂交的结果；间接ELISA法特异性鉴别试验证明特异性抗体对亲本株和减毒疫苗株(H2株)都有特异性的结合反应，证明获得的杂交瘤细胞株是分泌抗甲肝病毒特异性抗体的融合细胞株；对L-8株和H2株特异性鉴别试验也提示，也许此同一亚型的不同株的抗原决定簇非常相似，难以被特异性抗体所鉴别。
[Abstract]:Hepatitis A is an infectious disease that endangers human health. The pathogen is hepatitis A virus (Hepatitis A virus; HAV), belonging to a small RNA virus, only one serotype and multiple gene subtypes. Hepatitis A has been in different degrees of prevalence in developing countries for many years, the use of live attenuated and inactivated hepatitis a vaccine and hepatitis A The prevention provides a reliable means, but there are occasional cases of hepatitis A after vaccination, which requires the identification of the pathogens infected by the patients to make sure that the cause of the disease is the establishment of hybridoma technical preparation in the.1975 KOhler and Milstein cases caused by vaccination or hepatitis A virus infection. Monoclonal antibodies (monoclonal antibody; McAb). Monoclonal antibodies are specific antibodies against single antigen determinants. They are highly specific and are applied to the analysis of antigen relationships among different viruses or different strains of the same type. It has the advantages of strong specificity, high sensitivity, low cost and good reproducibility. Monoclonal antibodies were used to identify different strains of hepatitis A virus.
The hepatitis A virus L-8 strain is a strain isolated from the outbreak of hepatitis A epidemic in Shanghai in 1988. It belongs to the IB subtype. At present, the inactivated vaccine has been prepared and widely used in the country. We choose the hepatitis A virus L-8 strain related to our product as immunogen, immunized BALB / C mice, and the immunized spleen cells and myeloma cells (SP2 / 0). Through HAT selective culture system, the positive clones were screened by ELISA method, and the positive clones were cloned with limited dilution method. Finally, a hybridoma cell line that secreted the anti HAV specific antibody was screened. The titer of the supernatant and ascites was 1:64, 1:10000, and the chromosome of hybrid cells. The number was 80~90, which proved to be the result of two cell hybrids, and the indirect ELISA specific identification test showed that the specific antibody had specific binding reaction to the parent strain and the attenuated vaccine strain (H2 strain). It was proved that the hybridoma cell line obtained was a fusion cell line that secreted the specific anti - hepatitis C virus resistant body, and the specific discrimination against L-8 and H2 strains. Other tests also suggest that the epitopes of different strains of the same subtype are very similar and difficult to be identified by specific antibodies.

【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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