汉坦病毒核蛋白基因疫苗在小鼠体内表达动力学的初步研究

发布时间：2018-02-08 16:46

  本文关键词： 汉坦病毒 核酸疫苗 转染 组织分布 持续表达　出处：《中国医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：汉坦病毒核蛋白基因疫苗在小鼠体内表达动力学的初步研究 目的 肾综合症出血热(Hemorrhagic fever with renal syndrome,HFRS)是一种由汉坦病毒引起的急性病毒性传染病,起病急、症状重、病死率高,且目前尚无有效的治疗手段。因此,做好汉坦病毒的预防工作具有重要的现实意义。近年来,随着分子生物学的迅速发展,核酸疫苗作为一种新生物技术的出现为我们防治HFRS提供了新的思路,其相对于传统疫苗具有无可比拟的优势,代表者未来疫苗的发展方向。目前,已有研究者成功构建了若干亚型的汉坦病毒核酸疫苗,但研究重点更多的集中在核酸疫苗的免疫效果上,对于其在机体内的动力学研究相对较少。因此,本实验构建了含有汉坦病毒76-118株编码核蛋白(Neucleoprotein,NP)S抗原基因片段的核酸疫苗,研究其在体外真核细胞中的表达,肌肉接种后在小鼠体内组织分布及持续表达时间,进而探讨汉坦病毒核酸疫苗的应用前景。 本实验以此为目的,进行了以下研究:(1)以质粒pEGFP-C1为载体,质粒pTargeT/S中含有的S抗原基因片段为目的基因,构建GFP-NP融合蛋白真核表达重组质粒pEGFP/S;(2)脂质体体外转染Vero-E6细胞,观察重组质粒pEGFP/S在真核细胞中的表达;(3)免疫接种小鼠胫前肌,观察pEGFP/S在小鼠体内的组织分布及持续表达时间。 方法 一、汉坦病毒S基因真核表达重组体的构建 使用限制性内切酶EcoRI、Sa1 Ⅰ消化质粒pTargeT/S,纯化得到S基因片断,克隆至同样经EcoRI、Sa1 I消化的质粒pEGFP-C1上相应位点,酶切鉴定,得到重组质粒pEGFP/S。使用超纯质粒中提试剂盒提取质粒,-
[Abstract]:Preliminary study on the expression Kinetics of Hantavirus Nuclear protein Gene Vaccine in mice. Purpose. Hemorrhagic fever with renal syndrome (Hemorrhagic fever with renal syndrome HFRSs) is an acute viral infectious disease caused by Hantavirus. In recent years, with the rapid development of molecular biology, the emergence of nucleic acid vaccine as a new biotechnology provides us with a new idea for the prevention and treatment of HFRS. It has unparalleled advantages over traditional vaccines and represents the future direction of vaccine development. At present, researchers have successfully constructed several subtypes of Hantavirus nucleic acid vaccine. However, the focus of the study was on the immune effect of nucleic acid vaccine, and the kinetics of nucleic acid vaccine in organism was relatively little. Therefore, a nucleic acid vaccine containing nucleoprotein Neucleoprotein NPS gene fragment of Hantavirus 76-118 strain was constructed. To study the expression of Hantavirus in eukaryotic cells in vitro, the tissue distribution and duration of expression in mice after intramuscular inoculation, and to explore the application prospect of Hantavirus nucleic acid vaccine. For this purpose, the following studies were carried out on the following: 1. Using plasmid pEGFP-C1 as vector and S antigen gene fragment contained in plasmid pTargeT/S as target gene, the eukaryotic expression plasmid pEGFP / Sf-2) liposome of GFP-NP fusion protein was constructed and transfected into Vero-E6 cells in vitro. To observe the expression of recombinant plasmid pEGFP/S in eukaryotic cells and to observe the distribution and duration of pEGFP/S in the tibial anterior muscle of mice immunized. Method. 1. Construction of eukaryotic expression recombinant of Hantavirus S gene. The S gene fragment was purified by using restriction endonuclease EcoRIXSA1 鈪，

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