人呼吸道合胞病毒融合蛋白真核表达及纯化
发布时间：2018-11-13 11:43
【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染最重要的病毒病原。目前尚无特异性防治方法,世界卫生组织将发展RSV疫苗列为最优先发展的疫苗项目之一。在RSV所编码的11种蛋白中,融合蛋白(fusion protein, F)为跨膜糖蛋白,直接介导病毒与细胞的融合、病毒的穿入、合胞体的形成,而且F蛋白在RSV不同亚型间具有高度的抗原同源性,是主要的交叉保护性抗原,同时还有细胞毒性T淋巴细胞(CTL)识别的抗原表位。本研究尝试采用重组真核表达载体,瞬时转染COS-7细胞表达RSV F蛋白,利用操作简易、纯化效率高的Ni柱亲和层析法完成制备和纯化,获得高纯度、μg级的F蛋白,为进一步的诊断试剂及疫苗等研究奠定基础。 方法:根据编码F蛋白的基因序列设计并合成引物,以本实验室保存的含有F基因的质粒pGEM3zf-F为模板,利用PCR方法,扩增出3′端带His标签和凝血酶裂解位点的F基因序列,克隆入pGEM-T easy载体,经核酸序列分析后,进一步克隆到真核表达载体pcDNA3.1(+),限制性内切酶鉴定,用脂质体Lipofectamine 2000转染COS-7细胞,48 h后用RT-PCR鉴定基因转录,72 h后再用Western blot和间接免疫荧光检测目的蛋白的表达和分布。Ni柱亲和层析法纯化COS-7细胞表达的F蛋白,高效毛细管电泳分析纯化后蛋白纯度。 结果:核酸序列分析证实获得带His标签的RSV F基因序列,没有发生无义突变。真核表达载体pcDNA3.1(+)/F-His的限制性内切酶分析结果与预期一致。转染COS-7细胞后,RT-PCR检测到F基因有转录,Western blot分析观察到F蛋白特异性的表达条带,间接免疫荧光实验观察到F蛋白主要呈膜及胞浆分布。Ni柱法F-His蛋白纯化回收率约为14.1%,高效毛细管电泳分析显示纯化后的F蛋白纯度达99%以上。 结论:初步建立了RSV F蛋白真核表达及纯化方法,为进一步优化RSV F蛋白制备条件及单克隆抗体及诊断试剂等研究奠定了基础。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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相关期刊论文 前2条
1 吕治林;儿童疫苗的现状与展望[J];国外医学.流行病学传染病学分册;2000年01期
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