重组轮状病毒VP4抗原的表达及免疫原性研究

发布时间：2018-03-10 21:28

本文选题：轮状病毒VP4基因　切入点：重组腺病毒　出处：《中国协和医科大学》2005年博士论文　论文类型：学位论文

【摘要】：由于轮状病毒(Rotavirus，RV)引起的婴幼儿腹泻至今没有特效治疗药物，因此研制疫苗仍是预防该疾病的热点。目前轮状病毒疫苗的研制有减毒活疫苗，基因工程亚单位疫苗，遗传重组疫苗，核酸疫苗以及基因工程载体疫苗。无论何种形式的疫苗，其目的都是要刺激机体产生特异性免疫反应。作为消化道感染性疾病，特别希望能诱导局部分泌性免疫。本论文就轮状病毒重要中和抗原(Viral Protein 4，VP4)基因做了基因修饰，，及原核和真核系统的表达研究；对修饰后表达产物免疫原性进行了评价。 VP4是轮状病毒的外壳蛋白，具有血凝素、病毒吸附、决定病毒毒力等多种功能。我们的研究选择了轮状病毒SA11株VP4基因作为目的基因。SA11株的VP4基因长度为2363个碱基对(bp)，成熟蛋白有776个氨基酸(aa)，分子量为87kD左右。为了探讨提高抗原免疫原性的可能性，我们通过两种方式修饰了VP4基因。第一种修饰是在VP4基因的5′端引入来自于小鼠IgH链的分泌信号肽序列SG(139bp)，记为SG-VP4，目的是为了使野生型VP4基因获得糖基化；第二种修饰是除在5′端引入信号肽序列，还在VP4基因3′端引入来自人HLA Ⅰ类分子α链的3′端部分TM(750bp)，记为SG-VP4-TM，目的是使获得糖基化的VP4向胞外分泌，并停留于细胞膜，拟提高免疫原性。两种修饰基因都以基因组的构型引入，信号肽序列有一个内含子，而转膜基因含有两个内含子。表达之后，19个氨基酸的信号肽序列将被切除，而转膜基因的64个氨基酸将保留下来。表达之后，由于存在信号肽的作用，VP4被糖基化，分子量增至97kD左右。我们将修饰后的VP4基因克隆至穿梭质粒中，与腺病毒载体共转染
[Abstract]:Since there is no special therapeutic drug for infantile diarrhea caused by rotavirus Rotavirus RV, the development of vaccine is still a hot spot in the prevention of the disease. At present, there are attenuated live vaccine and genetic engineering subunit vaccine for rotavirus vaccine. Genetic recombinant vaccines, nucleic acid vaccines, and genetically engineered vector vaccines. Any form of vaccine is designed to stimulate a specific immune response in the body, as an infectious disease of the digestive tract. In this paper, the gene modification and expression of prokaryotic and eukaryotic system of the important neutralizing antigen virus Protein 4 / VP4 of rotavirus were studied, and the immunogenicity of the expressed product was evaluated. VP4 is a rotavirus coat protein with hemagglutinin, virus adsorption, We selected the VP4 gene of rotavirus SA11 strain as the target gene. SA11 strain, the length of VP4 gene is 2363 base pairs, the mature protein has 776 amino acids, the molecular weight is about 87kD. To explore the possibility of improving the immunogenicity of antigens, We modified the VP4 gene in two ways. The first modification was to introduce the secreting signal peptide sequence SGN139bpN from the mouse IgH chain into the 5'terminal of the VP4 gene, which was named SG-VP4, in order to glycosylation of the wild type VP4 gene. The second modification is the introduction of the signal peptide sequence at the 5'terminal, and the 3'terminal portion of the human HLA class I 伪 chain, called SG-VP4-TMN, at the 3'end of the VP4 gene. The purpose of the modification is to make the glycosylated VP4 secrete out of the cells and stay on the cell membrane. To improve immunogenicity, both modified genes were introduced into the genome, the signal peptide sequence had an intron, and the transmembrane gene contained two introns. After expression, the 19 amino acid signal peptide sequence would be removed. After expression, the molecular weight of VP4 was increased to about 97kD due to the glycosylation of VP4. The modified VP4 gene was cloned into shuttle plasmid and co-transfected with adenovirus vector.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392;Q789

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