人工合成SARS抗原基因CAG的克

发布时间：2018-05-02 16:55

本文选题：SARS冠状病毒 + S蛋白　；参考：《中国医科大学》2006年硕士论文

【摘要】：目的严重急性呼吸道综合征(severe acute respiratory syndrome,SARS)是一种新型急性传染性疾病,它的高死亡率严重威胁着人类的健康,在2004年仍有病例散发。其病原体是一种新的冠状病毒(SARS Coronavirus,SARS-CoV),来源于野生动物,SARS-CoV是单股正链RNA病毒,基因组全长约29.7kb,纤突蛋白(S)、囊膜蛋白(E)、基质蛋白(M)、及核衣壳蛋白(N)是它的主要结构蛋白。其中S蛋白是SARS-CoV主要的膜结合蛋白,为Ⅰ型跨膜糖蛋白,在致病过程中S蛋白是主要的毒力因子,在病毒吸附细胞受体、细胞融合及诱导中和抗体产生过程中发挥关键作用。为了预防SARS的再次流行,人们迫切需要一种安全有效的疫苗早日研制成功。目前有许多候选疫苗进入临床前研究,例如:灭活疫苗,DNA疫苗,多肽疫苗等。我们的实验是通过网络数据库结合生物信息学软件分析的方法,确定S蛋白中可能在SARS-CoV感染过程中起重要作用并具有较好抗原性参数的结构区域作为候选抗原表位,人工合成嵌合基因,体外表达后免疫家兔,为抗SARS基因工程疫苗的深入研究提供实验依据。方法 1.抗原表位预测运用生物信息学对SARS-CoV S蛋白的二级结构,亲水性,穿膜螺旋,N-糖基化位点,氨基酸序列的亲疏水性,表面可及性,分子刚性及抗原性参数进行预测分析,设计抗原表位多肽。 2.基因合成和测序鉴定设计串联多表位的嵌合基因,命名为CAG,全长1274 bp由大连TaKaRa公司合成及测序验证。 3.CAG嵌合基因的扩增
[Abstract]:Purpose Severe acute respiratory syndrome (SARS) is a new type of acute infectious disease. Its high mortality is a serious threat to human health. The pathogen of SARS-CoV is a new coronavirus, SARS-CoV. SARS-CoV is a single-strand positive strand RNA virus. Its genome length is about 29.7 kb. The main structural proteins of SARS-CoV are the envelope protein, matrix protein, nucleocapsid protein and nucleocapsid protein. Among them, S protein is the main membrane binding protein of SARS-CoV, which is type I transmembrane glycoprotein. S protein is the main virulence factor in the pathogenesis of SARS-CoV. It plays a key role in the process of virus adsorbing cell receptor, cell fusion and inducing neutralizing antibody. In order to prevent the re-prevalence of SARS, it is urgent to develop a safe and effective vaccine as soon as possible. At present, there are many candidate vaccines, such as inactivated DNA vaccine, polypeptide vaccine and so on. Through the method of network database and bioinformatics software analysis, we identified the structural regions of S protein that may play an important role in the process of SARS-CoV infection and have good antigenicity parameters as candidate epitopes. The chimeric gene was synthesized and expressed in vitro to immunize rabbits. It provides experimental basis for further study of anti SARS gene engineering vaccine. Method 1. Antigen epitope prediction The secondary structure, hydrophilicity, N-glycosylation site of SARS-CoV S protein, hydrophobicity, surface accessibility, molecular rigidity and antigenicity parameters of amino acid sequence were predicted and analyzed by bioinformatics, and antigen epitope peptides were designed. 2. Gene synthesis and sequencing A chimeric gene named CAG was designed and its length of 1274 BP was synthesized and sequenced by Dalian TaKaRa Company. Amplification of 3.CAG Chimeric Gene
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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