呼吸道合胞病毒F2蛋白亚单位疫苗的初步研究

发布时间：2018-07-15 09:55

【摘要】：呼吸道合胞病毒(Respiratory syncytial virus,RSV)是世界范围内普遍引起婴幼儿下呼吸道感染的主要病原体之一,几乎95%的幼儿在两岁之前都感染过RSV,还可以引起毛细支气管炎、肺炎、支气管炎。自从该病毒发现以来,虽然在RSV疫苗研制方面取得了一定效果,但目前还没有一种完全安全、有效的疫苗问世。本论文在原核细胞中表达了RSV的一种包膜蛋白F的F2肽段,并探索了所表达的F2蛋白的免疫原性。为了克隆F2蛋白基因和表达F2,在Hela细胞中培养RSV,病变达到80%以上时提取总RNA,用反转录PCR获得F2蛋白基因。将F2基因插入pGEX-6p-l载体,重组表达载体F2/pGEX-6p-l在大肠杆菌Rosetta中正确表达出了GST-F2蛋白,以包涵体的形式存在,表达量不足总蛋白的10%。通过对包涵体提取和洗涤,GST-F2蛋白可以占到50%。对包涵体变性、复性及使用GST亲和层析纯化,融合蛋白纯度达到85%。为了测定原核所表达的F2蛋白的免疫原性,36只实验小鼠分为6组,分别在0、2、4周免疫小鼠, A：PBS组；B：LT(大肠杆菌不耐热肠毒素,LT)组；C:GST组：D：GST+LT组；E：GST-F2组；F：GST-F2+LT组。分别在1、3、5周时采集血清,最后一次免疫后收集呼吸道灌洗液,通过ELISA测定血清IgG、IgG1、IgG2a、IgA和呼吸道粘膜分泌型IgA (sIgA)。结果表明,F组和E组的IgG、IgG1、IgG2a、IgA和sIgA水平显著高于其它对照组,说明融合蛋白可以刺激产生高的血清抗体和呼吸道粘膜抗体；E组的IgG2a水平高于F组,说明F2蛋白诱导的免疫应答主要为Th2型。F组和E组sIgA抗体显著高于其它对照组,二者之间没有显著差异,说明LT的粘膜佐剂效应没有明显表现。本文通过融合表达F2蛋白,纯化后与LT混合免疫小鼠,测定抗体水平可以看出F2蛋白有较好的免疫原性,而LT佐剂效应有限,这为以后研究F2蛋白在RSV中的致病机理和RSV疫苗研究及疫苗佐剂的选择方面奠定了初步的基础。
[Abstract]:Respiratory syncytial virus (RSV) is one of the major pathogens of infantile lower respiratory tract infection in the world. Almost 95% of children have been infected with RSVbefore the age of two years, and can also cause bronchiolitis, pneumonia, bronchitis. Since the discovery of the virus, although the RSV vaccine has achieved some results, there is not a completely safe and effective vaccine. In this paper, the F2 peptide of RSV, a kind of envelope protein F, was expressed in prokaryotic cells, and the immunogenicity of the expressed F2 protein was explored. In order to clone F2 gene and express F2, RSVs were cultured in Hela cells. Total RNAs were extracted when the lesion was over 80%. F2 protein gene was obtained by reverse transcription PCR. F2 gene was inserted into pGEX-6p-l vector. The recombinant expression vector F2 / pGEX-6p-l correctly expressed GST-F2 protein in Rosetta of Escherichia coli. The protein of GST-F _ 2 can account for 50% by extraction and washing of inclusion body. After denaturation, renaturation and purification by GST affinity chromatography, the purity of fusion protein reached 85%. In order to determine the immunogenicity of F2 protein expressed in prokaryotes, 36 experimental mice were divided into 6 groups. The mice were immunized in 0: 2 weeks, A: PBS group, B: LT group, C: GST group,% D: GST group, EW GST-F2 group, respectively. F: GST-F2 LT group. After the last immunization, the respiratory lavage fluid was collected. The serum IgG _ 1G _ (2) A and the secretory IgA (Siga) of the respiratory mucosa were measured by Elisa. The results were as follows: (1) after the last immunization, the serum IgG _ (1) and the secretory IgA (Siga) of the respiratory tract were determined by enzyme-linked immunosorbent assay (Elisa). The results showed that the levels of IgG2a and Siga in group F and group E were significantly higher than those in other control groups, indicating that the level of IgG2a in group E was higher than that in group F, which could stimulate the production of high serum antibody and respiratory mucosal antibody. The results showed that the immune response induced by F2 protein was significantly higher in Th2 type. F group and E group than in other control groups. There was no significant difference between the two groups, indicating that the mucosal adjuvant effect of LT was not obvious. After fusion and expression of F2 protein, mice were immunized with LT after purification. The antibody level showed that F2 protein had better immunogenicity, but the effect of LT adjuvant was limited. This will lay a foundation for further study on the pathogenesis of F2 protein in RSV and the study of RSV vaccine and the selection of vaccine adjuvant.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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