本文選題：輪狀病毒VP4基因　切入點：重組腺病毒　出處：《中國協(xié)和醫(yī)科大學》2005年博士論文　論文類型：學位論文

【摘要】：由于輪狀病毒(Rotavirus，RV)引起的嬰幼兒腹瀉至今沒有特效治療藥物，因此研制疫苗仍是預防該疾病的熱點。目前輪狀病毒疫苗的研制有減毒活疫苗，基因工程亞單位疫苗，遺傳重組疫苗，核酸疫苗以及基因工程載體疫苗。無論何種形式的疫苗，其目的都是要刺激機體產生特異性免疫反應。作為消化道感染性疾病，特別希望能誘導局部分泌性免疫。本論文就輪狀病毒重要中和抗原(Viral Protein 4，VP4)基因做了基因修飾，，及原核和真核系統(tǒng)的表達研究；對修飾后表達產物免疫原性進行了評價。 VP4是輪狀病毒的外殼蛋白，具有血凝素、病毒吸附、決定病毒毒力等多種功能。我們的研究選擇了輪狀病毒SA11株VP4基因作為目的基因。SA11株的VP4基因長度為2363個堿基對(bp)，成熟蛋白有776個氨基酸(aa)，分子量為87kD左右。為了探討提高抗原免疫原性的可能性，我們通過兩種方式修飾了VP4基因。第一種修飾是在VP4基因的5′端引入來自于小鼠IgH鏈的分泌信號肽序列SG(139bp)，記為SG-VP4，目的是為了使野生型VP4基因獲得糖基化；第二種修飾是除在5′端引入信號肽序列，還在VP4基因3′端引入來自人HLA Ⅰ類分子α鏈的3′端部分TM(750bp)，記為SG-VP4-TM，目的是使獲得糖基化的VP4向胞外分泌，并停留于細胞膜，擬提高免疫原性。兩種修飾基因都以基因組的構型引入，信號肽序列有一個內含子，而轉膜基因含有兩個內含子。表達之后，19個氨基酸的信號肽序列將被切除，而轉膜基因的64個氨基酸將保留下來。表達之后，由于存在信號肽的作用，VP4被糖基化，分子量增至97kD左右。 我們將修飾后的VP4基因克隆至穿梭質粒中，與腺病毒載體共轉染
[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 偽 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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R392;Q789

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