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  本文選題：甲肝病毒 + 雜交瘤細胞株　； 參考：《昆明醫(yī)學(xué)院》2006年碩士論文

【摘要】：甲型肝炎是危害人類健康的傳染病，其病原體為甲型肝炎病毒(Hepatitis A virus；HAV)，屬于小RNA病毒科毒粒，只有一個血清型和多個基因亞型。甲型肝炎在發(fā)展中國家多年來都有不同程度的流行，甲肝減毒活疫苗和滅活疫苗的使用，為甲肝的預(yù)防提供了可靠的手段，但偶有在接種疫苗后發(fā)生甲型肝炎的案例，這就需要對患者所感染的病原體進行鑒別，以明確患者發(fā)病的原因是接種疫苗引發(fā)還是甲肝野毒株感染導(dǎo)致的偶合病例。1975年KOhler和Milstein建立了雜交瘤技術(shù)制備了單克隆抗體(monoclonal antibody；McAb)。單克隆抗體是針對單一抗原決定簇的均一的特異的抗體，具有高度的特異性，被應(yīng)用于病毒不同型或同型不同株間的抗原關(guān)系分析，具有特異性強、靈敏度高，成本低，重復(fù)性好等優(yōu)點，因此，我們可以采用單克隆抗體對甲肝病毒的不同株進行鑒別。 甲肝病毒L-8株是我所在1988年上海甲肝暴發(fā)流行時分離出來的毒株，屬于IB亞型，目前已制備出滅活疫苗并在全國廣泛使用。我們選擇與我所產(chǎn)品相關(guān)的甲肝病毒L-8株作為免疫原，免疫BALB／C小鼠，取免疫后的脾細胞與骨髓瘤細胞(SP2／0)進行融合，通過HAT選擇性培養(yǎng)系統(tǒng)培養(yǎng)，ELISA法檢測抗體篩選陽性克隆，并對陽性克隆進行有限稀釋法克隆化培養(yǎng)，最后篩選出一株穩(wěn)定分泌抗HAV特異性抗體的雜交瘤細胞株，其上清和腹水效價分別為1：64；1：10000；雜交細胞染色體計數(shù)為80～90，，證明是兩種細胞雜交的結(jié)果；間接ELISA法特異性鑒別試驗證明特異性抗體對親本株和減毒疫苗株(H2株)都有特異性的結(jié)合反應(yīng)，證明獲得的雜交瘤細胞株是分泌抗甲肝病毒特異性抗體的融合細胞株；對L-8株和H2株特異性鑒別試驗也提示，也許此同一亞型的不同株的抗原決定簇非常相似，難以被特異性抗體所鑒別。
[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.

【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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