本文選題：免疫磁珠 + 人呼吸道合胞病毒�。� 參考：《安徽醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:人呼吸道合胞病毒(human respiratory syncytial virus,RSV)是造成世界范圍內(nèi)嬰幼兒下呼吸道病毒性感染最重要的病原。RSV融合(fusion glycoprotein, F)蛋白編碼基因變異小,在RSV的兩個(gè)亞型間具有高度的保守性,是主要的交叉保護(hù)性抗原和細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocytes, CTLs)的靶抗原。以純化的F蛋白制成的亞單位疫苗(PFP-1、PFP-2和PFP-3)對(duì)成人和兒童具有一定的保護(hù)作用,顯示其在疫苗研制中具有較好價(jià)值,同時(shí),純化的RSV F蛋白對(duì)開(kāi)發(fā)RSV血清學(xué)診斷試劑也具有重要意義。本文嘗試用免疫磁珠分離技術(shù)從可表達(dá)RSV F蛋白的重組腺病毒感染的HEK293細(xì)胞裂解液中純化F蛋白,試圖建立一種方便、簡(jiǎn)潔的純化F蛋白的方法。 方法:本文首先以RSV感染HEp-2細(xì)胞,待細(xì)胞出現(xiàn)病變(cytopathic effect, CPE)后,收獲細(xì)胞及培養(yǎng)液,離心,取上清,PEG6000沉淀,沉淀物經(jīng)溶解后通過(guò)蔗糖密度梯度離心純化。以純化的RSV作為抗原,免疫家兔制備RSV抗血清,經(jīng)純化后,獲得RSV多克隆抗體,并用此抗體包被磁性微球,并確定包被磁珠所需的抗體濃度和包被時(shí)間。用可表達(dá)RSV F的重組腺病毒FGAd/F感染293細(xì)胞,收獲細(xì)胞裂解液,利用包被好兔抗人RSV多克隆抗體的免疫磁珠富集和純化細(xì)胞裂解液中的F蛋白,同時(shí)建立夾心ELISA,檢測(cè)純化的F蛋白濃度以及F蛋白的回收率。 結(jié)果:獲得兔抗人RSV多克隆抗體,并包被磁性微球形成免疫磁珠,并利用免疫磁珠分離技術(shù)成功純化可表達(dá)RSV F的重組腺病毒FGAd/F感染293細(xì)胞裂解液中的F蛋白,經(jīng)SDS-PAGE,Western blot鑒定為分子量為145 kD-170 kD區(qū)間的F蛋白二聚體,再由夾心ELISA建立標(biāo)準(zhǔn)曲線(xiàn),檢測(cè)免疫磁珠分離技術(shù)純化的RSV F蛋白濃度為116μg/mL,成功的利用免疫磁珠分離技術(shù)從含有141μg的F蛋白的細(xì)胞裂解液中提取58μg的F蛋白,回收率為41.1%。 結(jié)論:本研究成功的利用免疫磁珠分離技術(shù)從可表達(dá)RSV F的重組腺病毒FGAd/F感染293細(xì)胞裂解液中提取F蛋白,此方法方便快速,減少了常規(guī)純化F蛋白繁瑣,昂貴等缺點(diǎn),也為制備F蛋白提供了一條新的思路。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R373

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