转基因枸杞表达HIV重组壳体蛋白的鉴定及其免疫原性的初步研究

发布时间：2018-03-22 02:01

本文选题：HIV-1　切入点：MA4-CA　出处：《山东省医学科学院》2006年硕士论文　论文类型：学位论文

【摘要】： 自1981年美国发现世界首例艾滋病患者,至今已死亡2000多万人,仅2003年新感染人数就有500万。目前全世界大约已有5800万人被感染,大约一半为15至24岁的青壮年。我国艾滋病病毒感染者迅猛增长,现已有84万人被感染HIV病毒,已经成为我国当前突出的卫生问题,目前还没有一种HIV疫苗问世,如何高效而迅捷的生产HIV疫苗成为关系人类生命健康的重大问题。应用转基因植物表达HIV疫苗,有其简洁可操作性优势。 HIV的壳体蛋白(CA)为病毒核心结构的主要蛋白,在病毒粒子中含量丰富,在HIV病毒的组装和成熟过程中起着至关重要的作用,在HIV的不同亚型间高度保守。野生型的CA以圆柱状颗粒存在,与4个HIV基质蛋白(MA)的氨基酸残基结合就能使CA的形状由圆柱状转至球状,进而增强CA的免疫原性。本研究利用有助于CA形成病毒样颗粒(VLP)结构的MA的最后四个氨基酸残基,与CA融合表达,形成的球状颗粒能够增强CA的免疫原性。枸杞与烟草同属于茄科植物,植物细胞易于遗传转化,安全性性高,利用其再生成植株的形成可进行目的基因的表达。植物细胞作为真核细胞表达体系,可对其表达产物进行糖基化、磷酸化等翻译后加工修饰和装配,从而有利于提高HIV CA疫苗的免疫原性。有报道利用转基因烟草表达的HIV-1的P24蛋白能保持较好的免疫原性。而枸杞作为一种宝贵的中药材,则富含多种对身体有益的成分;而其作为常年生植物其遗传性状更加稳定,所携带基因不易丢失。在没有相应的HIV疫苗问世以前,应用植物表达系统选择性大量安全地表达HIV抗原多肽及众多重组蛋白,研究它们的作用机理及其免疫活性,应对HIV病毒的高度变异性,以制备安全高效的HIV疫苗。我实验室与美国Utah大学的Dr. W.I. Sundquist, Professor合作将影响CA形成球状VLP结构的MA的四个核苷酸序列与CA融合形成MA4-CA,然后将源于水稻的一个富含甘氨酸序列的信号肽(GRP)连接在MA4-CA融合基因的3'端,从而使枸杞中表达的CA疫苗被引导进入根细胞间隙,并从根系不断地分泌到培养液中,由此产生出高纯度的CA疫苗。并在此基础上.进一步明确植物组织表达的CA重组疫苗的免疫原性。为利用转基因枸杞表达成本低廉,更为安全有效的HIV重组疫苗提供依据。研究目的: 利用本实验室构建好的分泌型植物表达载体,通过根癌农杆菌对枸杞进行遗传转化,筛选阳性克隆,从基因水平及蛋白水平鉴定转基因枸杞。应用所提取转基因枸杞蛋白免疫BALB/c小鼠,进行其免疫原性研究。以期应用转基因枸杞表
[Abstract]:More than 20 million people have died since 1981, when the world's first AIDS patient was found in the United States, with 5 million new infections in 2003 alone. So far, about 58 million people have been infected in the world. About half of them are young adults aged 15 to 24. The number of HIV-infected people in China has increased rapidly, and 840000 people have been infected with HIV virus, which has become a prominent health problem in our country, and no HIV vaccine has yet been introduced. How to produce HIV vaccine efficiently and quickly has become a major problem related to human life and health. The application of transgenic plants to express HIV vaccine has the advantages of simplicity and maneuverability. The shell protein of HIV is the main protein of the core structure of the virus. It is rich in virus particles and plays an important role in the assembly and maturation of HIV virus. The wild type CA exists as a cylindrical particle and binds to the amino acid residues of four HIV matrix proteins, which can change the shape of CA from a cylindrical to a spherical one. In order to enhance the immunogenicity of CA, the last four amino acid residues of MA, which is helpful for CA to form virus like VLPstructures, were fused with CA, and the immunogenicity of CA was enhanced by spherical particles. Both Lycium barbarum and tobacco belong to the family Solanaceae. The plant cells are easy to genetic transformation and high safety. The plant cells can be used as eukaryotic expression system to express the target gene. The expressed product can be modified and assembled by glycosylation, phosphorylation and other post-translational processing. It is reported that the P24 protein of HIV-1 expressed by transgenic tobacco can maintain good immunogenicity. As a valuable Chinese herbal medicine, Lycium barbarum is rich in many beneficial components. As perennial plants, their genetic traits are more stable and the genes they carry are not easily lost. In the absence of corresponding HIV vaccine, the plant expression system was used to selectively and safely express HIV antigenic peptides and many recombinant proteins in order to study their mechanism of action and their immunological activity, so as to deal with the high variability of HIV virus. In order to prepare a safe and efficient HIV vaccine, our laboratory will work with Dr. W. I. Sundquist of Utah University in the United States. Professor will affect the fusion of four nucleotide sequences of MA, which forms spherical VLP structure, with CA to form MA4-CA.Then it will be derived from rice, which is rich in Gan. The signal peptide of the amino acid sequence, GRP, is linked to the 3'end of the MA4-CA fusion gene. So that the CA vaccine expressed in Lycium barbarum is directed into the root cell gap and secreted from the root system into the culture medium. On the basis of this, the immunogenicity of CA recombinant vaccine expressed in plant tissues was further determined, which provided a basis for the use of transgenic Lycium barbarum to express HIV vaccine with low cost and more safety and effectiveness. Objectives of the study:. By using the secretory plant expression vector constructed in our laboratory, Lycium barbarum was transformed by Agrobacterium tumefaciens and positive clones were screened. Transgenic Lycium barbarum was identified from gene level and protein level. BALB/c mice were immunized with transgenic Lycium barbarum protein, and its immunogenicity was studied.
【学位授予单位】：山东省医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q943.2

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