特异性艾滋病核酸疫苗转导载体的构建及初步应用
发布时间：2018-07-28 07:31
【摘要】： 当下,在全世界对控制和预防艾滋病病毒(Human immunodeficiency virus,HIV)感染工作中尚无完全有效手段的前提下,要有效的预防并控制艾滋病病毒的感染,最佳的思路是开发出一种能够安全、有效的预防艾滋病病毒感染的疫苗。 本研究的目的是构建一种新型的具有特异性的基因转导载体TG蛋白。基于穿膜肽信号肽(TAT)、DNA识别及结合序列系统(GAL4/UAS)上,添加适当的纯化及鉴定标签His-tag,定向克隆得到编码TG蛋白的基因片段。以原核表达系统中的pET-28a为载体构建了并得到了原核表达重组质粒pET-TG。GAL4蛋白具有能够与UAS核苷酸序列特异性结合的能力,所以经过纯化的TG蛋白在一定条件下可在机体外与嵌入荧光蛋白基因EGFP片段的pVAX1-UAS-EGFP质粒特异性结合,然后再利用琼脂糖凝胶电泳实验就可以直观的证明TG蛋白可与pVAX1-UAS-EGFP质粒特异性结合。再摸索出TG蛋白与pVAX1-UAS-EGFP质粒结合活性最高时的质粒蛋白浓度和时间条件。在细胞水平上将蛋白与质粒特异性的结合物产物与293T细胞共浴,最后在荧光显微镜下通过对荧光的检测研究TG蛋白的细胞转导活性。 通过大肠杆菌表达系统表达分子量为21kDa的目的蛋白。通过琼脂糖凝胶电泳可以看出质粒与蛋白结合活性最强时质粒量为10μg,蛋白量为16μg,同时TG蛋白在10min内即可与pVAX1-UAS-EGFP结合,2h内其结合效率无明显变化。通过细胞转导发现pVAX1-UAS-EGFP质粒与TG蛋白结合的结合物转导293T细胞均出现特异性荧光,表明TAT基因具有穿膜作用的。本研究为艾滋病核酸疫苗转导载体研究的进一步深入提供依据。
[Abstract]:At present, under the premise that there is no completely effective means to prevent and control HIV infection in the world, the best way to prevent and control HIV infection is to develop a safe one. Effective vaccine against HIV infection. The aim of this study was to construct a novel gene transduction vector TG protein. Based on the transmembrane peptide signal peptide (TAT) DNA recognition and binding sequence system (GAL4/UAS), the appropriate purification and identification tag His-tagwas added, and the gene fragment encoding TG protein was cloned. Using pET-28a in prokaryotic expression system as a vector, the prokaryotic expression plasmid pET-TG.GAL4 protein has the ability to bind specifically to UAS nucleotide sequence. Therefore, the purified TG protein can specifically bind to the pVAX1-UAS-EGFP plasmid embedded in the EGFP fragment of the fluorescent protein gene outside the body under certain conditions. Then agarose gel electrophoresis was used to demonstrate that TG protein could bind specifically to pVAX1-UAS-EGFP plasmid. The concentration and time of TG protein binding to pVAX1-UAS-EGFP plasmid were found out. The protein and plasmide-specific conjugate product were combined with 293T cells at the cellular level. Finally, the cell transduction activity of TG protein was studied by fluorescence microscopy. The target protein with molecular weight of 21kDa was expressed by E. coli expression system. Agarose gel electrophoresis showed that the amount of plasmid was 10 渭 g and the amount of protein was 16 渭 g when the binding activity between plasmid and protein was the highest, and the binding efficiency of TG protein to pVAX1-UAS-EGFP could not change significantly within 2 hours after binding to pVAX1-UAS-EGFP in 10min. Specific fluorescence was found in 293T cells transduced by pVAX1-UAS-EGFP plasmid binding to TG protein, indicating that TAT gene has transmembrane effect. This study provides the basis for the further study of AIDS nucleic acid vaccine transduction vector.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
本文编号：2149362
[Abstract]:At present, under the premise that there is no completely effective means to prevent and control HIV infection in the world, the best way to prevent and control HIV infection is to develop a safe one. Effective vaccine against HIV infection. The aim of this study was to construct a novel gene transduction vector TG protein. Based on the transmembrane peptide signal peptide (TAT) DNA recognition and binding sequence system (GAL4/UAS), the appropriate purification and identification tag His-tagwas added, and the gene fragment encoding TG protein was cloned. Using pET-28a in prokaryotic expression system as a vector, the prokaryotic expression plasmid pET-TG.GAL4 protein has the ability to bind specifically to UAS nucleotide sequence. Therefore, the purified TG protein can specifically bind to the pVAX1-UAS-EGFP plasmid embedded in the EGFP fragment of the fluorescent protein gene outside the body under certain conditions. Then agarose gel electrophoresis was used to demonstrate that TG protein could bind specifically to pVAX1-UAS-EGFP plasmid. The concentration and time of TG protein binding to pVAX1-UAS-EGFP plasmid were found out. The protein and plasmide-specific conjugate product were combined with 293T cells at the cellular level. Finally, the cell transduction activity of TG protein was studied by fluorescence microscopy. The target protein with molecular weight of 21kDa was expressed by E. coli expression system. Agarose gel electrophoresis showed that the amount of plasmid was 10 渭 g and the amount of protein was 16 渭 g when the binding activity between plasmid and protein was the highest, and the binding efficiency of TG protein to pVAX1-UAS-EGFP could not change significantly within 2 hours after binding to pVAX1-UAS-EGFP in 10min. Specific fluorescence was found in 293T cells transduced by pVAX1-UAS-EGFP plasmid binding to TG protein, indicating that TAT gene has transmembrane effect. This study provides the basis for the further study of AIDS nucleic acid vaccine transduction vector.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
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相关期刊论文 前3条
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3 ;Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency[J];World Journal of Gastroenterology;2007年44期
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