免疫磁珠纯化人呼吸道合胞病毒融合蛋白方法的建立

发布时间：2018-06-14 23:54

本文选题：免疫磁珠 + 人呼吸道合胞病毒　；参考：《安徽医科大学》2011年硕士论文

【摘要】：目的:人呼吸道合胞病毒(human respiratory syncytial virus,RSV)是造成世界范围内婴幼儿下呼吸道病毒性感染最重要的病原。RSV融合(fusion glycoprotein, F)蛋白编码基因变异小,在RSV的两个亚型间具有高度的保守性,是主要的交叉保护性抗原和细胞毒性T淋巴细胞(cytotoxic T lymphocytes, CTLs)的靶抗原。以纯化的F蛋白制成的亚单位疫苗(PFP-1、PFP-2和PFP-3)对成人和儿童具有一定的保护作用,显示其在疫苗研制中具有较好价值,同时,纯化的RSV F蛋白对开发RSV血清学诊断试剂也具有重要意义。本文尝试用免疫磁珠分离技术从可表达RSV F蛋白的重组腺病毒感染的HEK293细胞裂解液中纯化F蛋白,试图建立一种方便、简洁的纯化F蛋白的方法。方法:本文首先以RSV感染HEp-2细胞,待细胞出现病变(cytopathic effect, CPE)后,收获细胞及培养液,离心,取上清,PEG6000沉淀,沉淀物经溶解后通过蔗糖密度梯度离心纯化。以纯化的RSV作为抗原,免疫家兔制备RSV抗血清,经纯化后,获得RSV多克隆抗体,并用此抗体包被磁性微球,并确定包被磁珠所需的抗体浓度和包被时间。用可表达RSV F的重组腺病毒FGAd/F感染293细胞,收获细胞裂解液,利用包被好兔抗人RSV多克隆抗体的免疫磁珠富集和纯化细胞裂解液中的F蛋白,同时建立夹心ELISA,检测纯化的F蛋白浓度以及F蛋白的回收率。结果:获得兔抗人RSV多克隆抗体,并包被磁性微球形成免疫磁珠,并利用免疫磁珠分离技术成功纯化可表达RSV F的重组腺病毒FGAd/F感染293细胞裂解液中的F蛋白,经SDS-PAGE,Western blot鉴定为分子量为145 kD-170 kD区间的F蛋白二聚体,再由夹心ELISA建立标准曲线,检测免疫磁珠分离技术纯化的RSV F蛋白浓度为116μg/mL,成功的利用免疫磁珠分离技术从含有141μg的F蛋白的细胞裂解液中提取58μg的F蛋白,回收率为41.1%。结论:本研究成功的利用免疫磁珠分离技术从可表达RSV F的重组腺病毒FGAd/F感染293细胞裂解液中提取F蛋白,此方法方便快速,减少了常规纯化F蛋白繁琐,昂贵等缺点,也为制备F蛋白提供了一条新的思路。
[Abstract]:Objective: human respiratory syncytial virus (HRV) is the most important pathogen of infantile lower respiratory tract virus infection in the world. RSV fusion glycoprotein (FV) protein coding gene has little variation and is highly conserved between the two subtypes of RSV. It is the main cross-protective antigen and the target antigen of cytotoxic T lymphocytes (CTLs). PFP-1 and PFP-3), a subunit vaccine prepared from purified F protein, have protective effects on both adults and children, indicating that PFP-1 and PFP-3 have good value in vaccine development. The purified RSV F protein is also important for the development of RSV serological diagnostic reagent. This paper attempts to purify F protein from HEK293 cell lysate infected with recombinant adenovirus which can express RSV F protein by immunomagnetic bead technique, and try to establish a simple and convenient method for purifying F protein. Methods: HEp-2 cells were infected with RSV at first. After cytopathic effect (CPE) appeared in the cells, the cells and culture medium were harvested and centrifuged. The supernatant PEG6000 was precipitated. The precipitate was purified by sucrose density gradient centrifugation after dissolution. The antiserum of RSV was prepared by immunizing rabbits with purified RSV as antigen. After purification, the polyclonal antibody of RSV was obtained. The antibody was coated with magnetic microspheres, and the concentration of antibody and the time of coating were determined. The recombinant adenovirus FGAd-F expressing RSV was used to infect 293 cells and harvested cell lytic fluid. The F protein was enriched and purified by immunomagnetic beads coated with rabbit polyclonal antibody against RSV. At the same time, sandwich Elisa was established to detect the concentration of purified F protein and the recovery rate of F protein. Results: rabbit anti-human RSV polyclonal antibody was obtained and coated with magnetic microspheres to form immunomagnetic beads. The recombinant adenovirus FGAd-F expressing RSV F was successfully purified by immunomagnetic bead separation technique. The F protein dimer with a molecular weight of 145kD-170kD was identified by SDS-PAGEG blot, and the standard curve was established by sandwich Elisa. The concentration of RSV F protein purified by immunomagnetic bead separation technique was 116 渭 g / mL. 58 渭 g F protein was extracted from the cell lysate containing 141 渭 g F protein by immunomagnetic beads separation technique. The recovery rate was 41.1%. Conclusion: in this study, F protein was extracted successfully from 293 cell lysate infected by recombinant adenovirus FGAd-F expressing RSV F by immunomagnetic bead separation technique. This method is convenient and rapid, and reduces the disadvantages of routine purification of F protein, such as tedious and expensive. It also provides a new idea for the preparation of F protein.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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