發(fā)布時間：2018-02-08 16:46

  本文關(guān)鍵詞： 漢坦病毒 核酸疫苗 轉(zhuǎn)染 組織分布 持續(xù)表達(dá)　出處：《中國醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：漢坦病毒核蛋白基因疫苗在小鼠體內(nèi)表達(dá)動力學(xué)的初步研究 目的 腎綜合癥出血熱(Hemorrhagic fever with renal syndrome,HFRS)是一種由漢坦病毒引起的急性病毒性傳染病,起病急、癥狀重、病死率高,且目前尚無有效的治療手段。因此,做好漢坦病毒的預(yù)防工作具有重要的現(xiàn)實意義。近年來,隨著分子生物學(xué)的迅速發(fā)展,核酸疫苗作為一種新生物技術(shù)的出現(xiàn)為我們防治HFRS提供了新的思路,其相對于傳統(tǒng)疫苗具有無可比擬的優(yōu)勢,代表者未來疫苗的發(fā)展方向。目前,已有研究者成功構(gòu)建了若干亞型的漢坦病毒核酸疫苗,但研究重點更多的集中在核酸疫苗的免疫效果上,對于其在機體內(nèi)的動力學(xué)研究相對較少。因此,本實驗構(gòu)建了含有漢坦病毒76-118株編碼核蛋白(Neucleoprotein,NP)S抗原基因片段的核酸疫苗,研究其在體外真核細(xì)胞中的表達(dá),肌肉接種后在小鼠體內(nèi)組織分布及持續(xù)表達(dá)時間,進(jìn)而探討漢坦病毒核酸疫苗的應(yīng)用前景。 本實驗以此為目的,進(jìn)行了以下研究:(1)以質(zhì)粒pEGFP-C1為載體,質(zhì)粒pTargeT/S中含有的S抗原基因片段為目的基因,構(gòu)建GFP-NP融合蛋白真核表達(dá)重組質(zhì)粒pEGFP/S;(2)脂質(zhì)體體外轉(zhuǎn)染Vero-E6細(xì)胞,觀察重組質(zhì)粒pEGFP/S在真核細(xì)胞中的表達(dá);(3)免疫接種小鼠脛前肌,觀察pEGFP/S在小鼠體內(nèi)的組織分布及持續(xù)表達(dá)時間。 方法 一、漢坦病毒S基因真核表達(dá)重組體的構(gòu)建 使用限制性內(nèi)切酶EcoRI、Sa1 Ⅰ消化質(zhì)粒pTargeT/S,純化得到S基因片斷,克隆至同樣經(jīng)EcoRI、Sa1 I消化的質(zhì)粒pEGFP-C1上相應(yīng)位點,酶切鑒定,得到重組質(zhì)粒pEGFP/S。使用超純質(zhì)粒中提試劑盒提取質(zhì)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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