人呼吸道合胞病毒融合蛋白真核表達及純化
發(fā)布時間：2018-11-13 11:43
【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)廣泛分布于世界各地,是導(dǎo)致嬰幼兒嚴重下呼吸道感染最重要的病毒病原。目前尚無特異性防治方法,世界衛(wèi)生組織將發(fā)展RSV疫苗列為最優(yōu)先發(fā)展的疫苗項目之一。在RSV所編碼的11種蛋白中,融合蛋白(fusion protein, F)為跨膜糖蛋白,直接介導(dǎo)病毒與細胞的融合、病毒的穿入、合胞體的形成,而且F蛋白在RSV不同亞型間具有高度的抗原同源性,是主要的交叉保護性抗原,同時還有細胞毒性T淋巴細胞(CTL)識別的抗原表位。本研究嘗試采用重組真核表達載體,瞬時轉(zhuǎn)染COS-7細胞表達RSV F蛋白,利用操作簡易、純化效率高的Ni柱親和層析法完成制備和純化,獲得高純度、μg級的F蛋白,為進一步的診斷試劑及疫苗等研究奠定基礎(chǔ)。 方法:根據(jù)編碼F蛋白的基因序列設(shè)計并合成引物,以本實驗室保存的含有F基因的質(zhì)粒pGEM3zf-F為模板,利用PCR方法,擴增出3′端帶His標簽和凝血酶裂解位點的F基因序列,克隆入pGEM-T easy載體,經(jīng)核酸序列分析后,進一步克隆到真核表達載體pcDNA3.1(+),限制性內(nèi)切酶鑒定,用脂質(zhì)體Lipofectamine 2000轉(zhuǎn)染COS-7細胞,48 h后用RT-PCR鑒定基因轉(zhuǎn)錄,72 h后再用Western blot和間接免疫熒光檢測目的蛋白的表達和分布。Ni柱親和層析法純化COS-7細胞表達的F蛋白,高效毛細管電泳分析純化后蛋白純度。 結(jié)果:核酸序列分析證實獲得帶His標簽的RSV F基因序列,沒有發(fā)生無義突變。真核表達載體pcDNA3.1(+)/F-His的限制性內(nèi)切酶分析結(jié)果與預(yù)期一致。轉(zhuǎn)染COS-7細胞后,RT-PCR檢測到F基因有轉(zhuǎn)錄,Western blot分析觀察到F蛋白特異性的表達條帶,間接免疫熒光實驗觀察到F蛋白主要呈膜及胞漿分布。Ni柱法F-His蛋白純化回收率約為14.1%,高效毛細管電泳分析顯示純化后的F蛋白純度達99%以上。 結(jié)論:初步建立了RSV F蛋白真核表達及純化方法,為進一步優(yōu)化RSV F蛋白制備條件及單克隆抗體及診斷試劑等研究奠定了基礎(chǔ)。
[Abstract]:Objective: human respiratory syncytial virus (Human Respiratory Syncytial Virus,RSV) is one of the most important viral pathogens in infants with severe lower respiratory tract infection. At present, there is no specific method of prevention and treatment. The World Health Organization (WHO) has made the development of RSV vaccine one of the top priority vaccine projects. Among the 11 proteins encoded by RSV, the fusion protein (fusion protein, F) is a transmembrane glycoprotein that directly mediates the fusion of viruses with cells, the penetration of viruses, and the formation of syncytial bodies. Moreover, F protein has high antigenic homology among different subtypes of RSV, which is the main cross-protective antigen, and also has antigen epitopes recognized by (CTL) of cytotoxic T lymphocytes. In this study, the recombinant eukaryotic expression vector was used to express RSV F protein by transient transfection of COS-7 cells. The high purity, 渭 g F protein was obtained by Ni column affinity chromatography with high purification efficiency and simple operation. To lay a foundation for the further study of diagnostic reagents and vaccines. Methods: according to the gene sequence of F protein, primers were designed and synthesized. Using plasmid pGEM3zf-F containing F gene preserved in our laboratory as template, the F gene sequence with His tag and thrombin cleavage site was amplified by PCR. The plasmid was cloned into pGEM-T easy vector. After nucleic acid sequence analysis, the eukaryotic expression vector pcDNA3.1 (), restriction endonuclease was further cloned and transfected into COS-7 cells by liposome Lipofectamine 2000. After 48 h, the gene transcription was identified by RT-PCR. After 72 h, the expression and distribution of the target protein were detected by Western blot and indirect immunofluorescence. The F protein expressed in COS-7 cells was purified by Ni affinity chromatography, and the purity of the purified protein was analyzed by high performance capillary electrophoresis (HPCE). Results: nucleic acid sequence analysis confirmed that RSV F gene sequence with His tag had no nonsense mutation. The results of restriction endonuclease analysis of eukaryotic expression vector pcDNA3.1 () / F-His were consistent with expectations. After transfection of COS-7 cells, RT-PCR detected that F gene had transcriptional, Western blot analysis and observed the specific expression band of F protein. Indirect immunofluorescence assay showed that F protein mainly distributed in membrane and cytoplasm. The recovery rate of F-His protein purified by Ni column method was about 14. 1%, and the purity of purified protein was over 99% by high performance capillary electrophoresis. Conclusion: the method of eukaryotic expression and purification of RSV F protein was established, which laid a foundation for further optimization of preparation conditions, monoclonal antibodies and diagnostic reagents of RSV F protein.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
本文編號：2329010
[Abstract]:Objective: human respiratory syncytial virus (Human Respiratory Syncytial Virus,RSV) is one of the most important viral pathogens in infants with severe lower respiratory tract infection. At present, there is no specific method of prevention and treatment. The World Health Organization (WHO) has made the development of RSV vaccine one of the top priority vaccine projects. Among the 11 proteins encoded by RSV, the fusion protein (fusion protein, F) is a transmembrane glycoprotein that directly mediates the fusion of viruses with cells, the penetration of viruses, and the formation of syncytial bodies. Moreover, F protein has high antigenic homology among different subtypes of RSV, which is the main cross-protective antigen, and also has antigen epitopes recognized by (CTL) of cytotoxic T lymphocytes. In this study, the recombinant eukaryotic expression vector was used to express RSV F protein by transient transfection of COS-7 cells. The high purity, 渭 g F protein was obtained by Ni column affinity chromatography with high purification efficiency and simple operation. To lay a foundation for the further study of diagnostic reagents and vaccines. Methods: according to the gene sequence of F protein, primers were designed and synthesized. Using plasmid pGEM3zf-F containing F gene preserved in our laboratory as template, the F gene sequence with His tag and thrombin cleavage site was amplified by PCR. The plasmid was cloned into pGEM-T easy vector. After nucleic acid sequence analysis, the eukaryotic expression vector pcDNA3.1 (), restriction endonuclease was further cloned and transfected into COS-7 cells by liposome Lipofectamine 2000. After 48 h, the gene transcription was identified by RT-PCR. After 72 h, the expression and distribution of the target protein were detected by Western blot and indirect immunofluorescence. The F protein expressed in COS-7 cells was purified by Ni affinity chromatography, and the purity of the purified protein was analyzed by high performance capillary electrophoresis (HPCE). Results: nucleic acid sequence analysis confirmed that RSV F gene sequence with His tag had no nonsense mutation. The results of restriction endonuclease analysis of eukaryotic expression vector pcDNA3.1 () / F-His were consistent with expectations. After transfection of COS-7 cells, RT-PCR detected that F gene had transcriptional, Western blot analysis and observed the specific expression band of F protein. Indirect immunofluorescence assay showed that F protein mainly distributed in membrane and cytoplasm. The recovery rate of F-His protein purified by Ni column method was about 14. 1%, and the purity of purified protein was over 99% by high performance capillary electrophoresis. Conclusion: the method of eukaryotic expression and purification of RSV F protein was established, which laid a foundation for further optimization of preparation conditions, monoclonal antibodies and diagnostic reagents of RSV F protein.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前2條
1 呂治林;兒童疫苗的現(xiàn)狀與展望[J];國外醫(yī)學.流行病學傳染病學分冊;2000年01期
2 董繼華,盧銀平,江漢珍,田順新;炭凝集試驗快速檢測病毒的研究[J];細胞與分子免疫學雜志;2001年05期
，本文編號：2329010
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2329010.html
最近更新
教材專著
