以改造隐性表位为基础的HIV复合多表位疫苗的构建及其免疫应答分析

发布时间：2018-05-03 07:01

本文选题：HIV-1 + 隐性表位　；参考：《第四军医大学》2010年硕士论文

【摘要】： 艾滋病(AIDS)在世界上流行已经将近30年了，到目前为止，人类无论是在治疗上还是在预防上都没有找到彻底消灭HIV的方法，因此，研究有效的预防性或治疗性疫苗迫在眉睫，用以防止HIV感染以及减少AIDS的发病。 CD8+T细胞在HIV感染早期发挥重要作用，但是由于HIV在压力选择下容易发生突变，可以逃避特异性的CTL反应，，而HIV-1 gag、pol序列是其重要的蛋白组成部分，具有相对保守的序列，选择其中的表位可能突破因高突变导致的免疫清除这一障碍。本研究旨在探索以改造隐性表位为基础，同时选择具有颗粒样结构的HIV p24蛋白作为外源抗原支架载体系统，依据其氨基酸序列构建HIV复合多CTL表位基因，制备核酸及蛋白疫苗，免疫转基因动物，激发特异性CTL反应。传统上对低亲和力表位有一定忽视，修饰低MHC亲和力表位可以突破性提高其免疫原性，并且诱导发生强烈而耐受突变的CD8+T细胞特异性免疫反应，为研制安全有效的HIV预防/治疗性疫苗提供新途径。 DNA疫苗是含有编码目的抗原蛋白基因的双链DNA分子(如质粒)，将其直接注射到体内，可以通过体内细胞表达相应的抗原蛋白诱导相应的免疫反应。将构建的HIV复合多CTL表位克隆入真核表达载体pcDNA3.1(+)，同过转化大肠杆菌XL-10并扩菌，通过大提质粒，获得能够达到一定剂量的含有目的基因的DNA疫苗。将构建好的HIV复合多CTL表位克隆入原核表达载体pET32a，转化大肠杆菌BL-21，IPTG诱导，发现有目的蛋白的过表达，采用包涵体纯化的方法对目的蛋白经行纯化，因为是在变性条件下纯化的蛋白，还需要对蛋白进行复性，以使得蛋白更接近天然状态下的活性和结构，这样就制备了蛋白亚单位疫苗。本研究选择的实验动物为BALA/c小鼠和HHD-2小鼠，按照单一DNA疫苗、单一蛋白疫苗、单一重组腺病毒疫苗(重组腺病毒疫苗由本室李英辉博士构建，本文不再赘述)以及复合疫苗免疫的方案对上述小鼠进行免疫。通过检测抗体以及细胞因子、细胞毒等体液和细胞免疫两个方面来分析各种疫苗的免疫效果。实验结果提示HIV复合多CTL表位疫苗能够诱导动物体内的细胞和体液免疫应答。本研究实验结果为探索新型的抗HIV-1疫苗提供了新依据。
[Abstract]:AIDS ( AIDS ) has been epidemic in the world for nearly 30 years . So far , human beings have not found a way to completely eradicate HIV , both in treatment and in prevention , so the study of effective preventive or therapeutic vaccines is urgent to prevent HIV infection and reduce the incidence of AIDS .

CD8 + T cells play an important role in the early stage of HIV infection , but HIV - 1 gag and pol sequences are important protein components , and HIV - 1 gag and pol sequences are important protein components . HIV - 1 gag and pol sequences have relatively conserved sequences .

The low affinity epitope is traditionally ignored , and the modified low MHC affinity epitope can improve the immunogenicity and induce a strong and tolerant CD8 + T cell - specific immune response to provide a new path for the development of a safe and effective HIV prevention / therapeutic vaccine .

The DNA vaccine is a double - stranded DNA molecule ( such as a plasmid ) containing an antigen protein gene encoding a target , can be directly injected into the body , and the corresponding immune response can be induced by the expression of the corresponding antigen protein by in vivo cells .

The constructed HIV complex multi - CTL epitope is cloned into a prokaryotic expression vector pET32a , transformed into E . coli BL - 21 , induced by IPTG to find the over - expression of the target protein , and the target protein is purified by inclusion body purification , because the protein is purified under denaturing conditions , and the protein is required to be restored , so that the protein is closer to the activity and the structure in the natural state , so that the protein subunit vaccine is prepared .

BALB / c mice and HHD - 2 mice were immunized with single DNA vaccine , single protein vaccine , single recombinant adenovirus vaccine ( the recombinant adenovirus vaccine was constructed by Dr . Lee Ying - hui , our lab ) . The immune effects of various vaccines were analyzed by detecting antibodies , cytokines , cytotoxic and humoral and cellular immunity . The results suggested that the HIV complex CTL epitope vaccine could induce cellular and humoral immune responses in animals .

The results of this study provide a new basis for exploring new anti - HIV - 1 vaccine .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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