呼吸道合胞病毒重组融合蛋白F2的免疫原性研究

发布时间：2018-01-09 08:17

本文关键词：呼吸道合胞病毒重组融合蛋白F2的免疫原性研究　出处：《昆明理工大学》2011年硕士论文　论文类型：学位论文

【摘要】：呼吸道合胞病毒是引起婴幼儿下呼吸道感染的主要病原体,对婴幼儿健康造成很大威胁。已研制的RSV疫苗在使用中常常会出现Th2型免疫极化现象和免疫病理损伤,本文将呼吸道合胞病毒F2蛋白与结核分枝杆菌ESAT-6蛋白在大肠杆菌中融合表达,并与LT佐剂共同免疫,诱导机体产生Th1/Th2平衡免疫反应的同时,在呼吸道产生粘膜保护性抗体。本论文将呼吸道合胞病毒F2蛋白与结核分枝杆菌ESAT-6蛋白的基因用PCR方法连接在一起,在大肠杆菌中经IPTG诱导后,ESAT-6-F2和ESAT-6蛋白均以包涵体形式高效表达,分子量分别为21kDa和10kDa,免疫印迹法证明表达的蛋白为ESAT-6-F2和ESAT-6,尿素变性后用镍离子亲和层析法进行纯化,纯度均达到90%以上。最后通过梯度透析复性成功复性于PBS缓冲液中。免疫共分5组,分别为：PBS组、LT组、ESAT-6组、ESAT-6-F2+LT组、ESAT-6-F2组,每组5只小鼠。实验结果显示,ESAT-6-F2+LT组、ESAT-6-F2组和ESAT-6组总IgG抗体效价比PBS高(P0.05),说明两个蛋白免疫原性较好,都倾向于Th2型免疫反应,而LT佐剂的加入削弱了ESAT-6-F2蛋白的免疫极化现象。同批免疫的小鼠末次免疫两周后开始攻毒,连续三天,剂量为每天每只小鼠攻毒5×105pfu,以未攻毒小鼠作为空白对照。结果表明,攻毒后小鼠体重均大幅下降,ESAT-6-F2+LT组小鼠减重明显少于PBS组,说明ESAT-6-F2蛋白与LT共同免疫,对小鼠产生一定的保护效果。抗体检测和肺部病理切片结果显示,ESAT-6组Th2型免疫极化现象加剧,肺部病变严重,而ESAT-6-F2+LT诱导较为平衡的免疫应答,肺部病变较轻,安全性较好。本论文初步评价了ESAT-6-F2蛋白的免疫原性,为呼吸道合胞病毒疫苗研制奠定基础。
[Abstract]:Respiratory syncytial virus (RSV) is the main pathogen of infantile lower respiratory tract infection. The RSV vaccine that has been developed is often used in the use of Th2 type immune polarization phenomenon and immune pathological damage. In this paper, respiratory syncytial virus F2 protein and Mycobacterium tuberculosis ESAT-6 protein were expressed in E. coli and immunized with LT adjuvant. While inducing Th1/Th2 balanced immune response, mucosal protective antibodies were produced in respiratory tract. In this paper, the gene of respiratory syncytial virus F2 and ESAT-6 protein of Mycobacterium tuberculosis were linked together by PCR method and induced by IPTG in Escherichia coli. ESAT-6-F2 and ESAT-6 proteins were highly expressed in the form of inclusion bodies with molecular weight of 21kDa and 10kDa, respectively. The protein was identified as ESAT-6-F2 and ESAT-6 by Western blot and purified by nickel ion affinity chromatography after urea denaturation. The purity was above 90%. Finally, the renaturation was successfully refolded in PBS buffer by gradient dialysis. The immunized mice were divided into 5 groups respectively. The mice were divided into 5 groups: ESAT-6, ESAT-6-F2, ESAT-6-F2, respectively. The total IgG antibody titer of ESAT-6-F2 group and ESAT-6 group in ESAT-6-F2 LT group was higher than that in PBS group, indicating that the immunogenicity of the two proteins was better. LT adjuvant weakened the immune polarization of ESAT-6-F2 protein. The mice immunized with the same batch of mice began to attack the virus two weeks after the last immunization for three consecutive days. The dose was 5 脳 10 5 pfux per mouse per day. The mice without the virus were used as the blank control. The weight loss of ESAT-6-F2 LT group was significantly lower than that of PBS group, indicating that ESAT-6-F2 protein and LT co-immunized. The results of antibody detection and pathological section of lung showed that the Th2 type immunoreactive phenomenon in ESAT-6 group was aggravated and the lung lesion was serious. ESAT-6-F2 LT induced a more balanced immune response with mild pulmonary lesions and better safety. In this paper, the immunogenicity of ESAT-6-F2 protein was preliminarily evaluated, which laid a foundation for the development of respiratory syncytial virus vaccine.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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