呼吸道合胞病毒F2蛋白亞單位疫苗的初步研究
發(fā)布時(shí)間：2018-07-15 09:55
【摘要】：呼吸道合胞病毒(Respiratory syncytial virus,RSV)是世界范圍內(nèi)普遍引起嬰幼兒下呼吸道感染的主要病原體之一,幾乎95%的幼兒在兩歲之前都感染過RSV,還可以引起毛細(xì)支氣管炎、肺炎、支氣管炎。自從該病毒發(fā)現(xiàn)以來,雖然在RSV疫苗研制方面取得了一定效果,但目前還沒有一種完全安全、有效的疫苗問世。 本論文在原核細(xì)胞中表達(dá)了RSV的一種包膜蛋白F的F2肽段,并探索了所表達(dá)的F2蛋白的免疫原性。 為了克隆F2蛋白基因和表達(dá)F2,在Hela細(xì)胞中培養(yǎng)RSV,病變達(dá)到80%以上時(shí)提取總RNA,用反轉(zhuǎn)錄PCR獲得F2蛋白基因。將F2基因插入pGEX-6p-l載體,重組表達(dá)載體F2/pGEX-6p-l在大腸桿菌Rosetta中正確表達(dá)出了GST-F2蛋白,以包涵體的形式存在,表達(dá)量不足總蛋白的10%。通過對(duì)包涵體提取和洗滌,GST-F2蛋白可以占到50%。對(duì)包涵體變性、復(fù)性及使用GST親和層析純化,融合蛋白純度達(dá)到85%。 為了測(cè)定原核所表達(dá)的F2蛋白的免疫原性,36只實(shí)驗(yàn)小鼠分為6組,分別在0、2、4周免疫小鼠, A：PBS組；B：LT(大腸桿菌不耐熱腸毒素,LT)組；C:GST組：D：GST+LT組；E：GST-F2組；F：GST-F2+LT組。分別在1、3、5周時(shí)采集血清,最后一次免疫后收集呼吸道灌洗液,通過ELISA測(cè)定血清IgG、IgG1、IgG2a、IgA和呼吸道粘膜分泌型IgA (sIgA)。 結(jié)果表明,F組和E組的IgG、IgG1、IgG2a、IgA和sIgA水平顯著高于其它對(duì)照組,說明融合蛋白可以刺激產(chǎn)生高的血清抗體和呼吸道粘膜抗體；E組的IgG2a水平高于F組,說明F2蛋白誘導(dǎo)的免疫應(yīng)答主要為Th2型。F組和E組sIgA抗體顯著高于其它對(duì)照組,二者之間沒有顯著差異,說明LT的粘膜佐劑效應(yīng)沒有明顯表現(xiàn)。 本文通過融合表達(dá)F2蛋白,純化后與LT混合免疫小鼠,測(cè)定抗體水平可以看出F2蛋白有較好的免疫原性,而LT佐劑效應(yīng)有限,這為以后研究F2蛋白在RSV中的致病機(jī)理和RSV疫苗研究及疫苗佐劑的選擇方面奠定了初步的基礎(chǔ)。
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.1
本文編號(hào)：2123689
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.1
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相關(guān)期刊論文 前5條
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