含塞姆利基森林病毒复制子的HIV多表位核酸疫苗的构建与表达

发布时间：2018-05-23 20:35

本文选题：HIV + 塞姆利基森林病毒复制子　；参考：《延边大学》2007年硕士论文

【摘要】： 获得性免疫缺陷综合征(Acquired immunodeficiency syndrome，AIDS)即艾滋病，是由人免疫缺陷病毒(Human jmmunodeficiency Virus，HIV)感染引起的一种恶性传染病。自从1981年在美国首次发现该病以来，全球已有6 500多万感染病例，艾滋病还在以惊人的速度蔓延着，，严重威胁人类的健康。HIV基因高度变异，逃避免疫监视，使得传统疫苗较难产生有效的免疫保护。核酸疫苗是从基因治疗领域发展起来的一种全新的免疫防治剂，可同时诱导细胞免疫应答和体液免疫应答，安全性好，构建灵活，成为一种非常有希望的艾滋病候选疫苗。 pSFV1载体为衍生于塞姆利基森林病毒(Semliki forest virus，SFV)RNA复制子的真核表达载体，但此载体系统应用时需在体外将质粒DNA转录成RNA，操作很不方便，限制了其广泛应用。为此，本研究通过分子克隆技术，将SFV的复制子插入到真核表达载体pIRESneo的CMVie立即早期启动子和腺苷酸转录终止信号Poly(A)之间，构建了含塞姆利基森林病毒复制子的DNA表达载体pCS，改造后的载体可按常规DNA疫苗的方式进行制备、免疫，克服了原载体pSFV1体外转录制备RNA的弊端。将增强型绿色荧光蛋白(EGFP)基因片段插入到pCS载体多克隆位点中，得到pCS-EGFP，用其转染BHK-21细胞，荧光显微镜下能观察到EGFP的表达产物增强型绿色荧光蛋白，表明该新型表达载体可以用于表达外源基因。为了探讨此新型载体pCS用于研制抗病毒疫苗的可能性，本研究选择HIV优势抗原表位，以HIV-1表位基因为基础，对抗原基因进行人工分子设计，将HIV多表位基因(Multiple-epitope gene，MEG)与HIV-1巨分子颗粒p24进行嵌合。从已构建保存的pVAX1-MEGp24酶切下HIV嵌合多表位MEGp24基因，克隆到pCS表达载体中，成功构建了含塞姆利基森林病毒复制子的HIV多表位核酸疫苗pCS-MEGp24。间接免疫荧光试验(IFA)检测表明MEGp24基因在转染BHK-21细胞中得到有效表达，RT-PCR可检测到MEGp24基因的转录产物mRNA。综上所述，本试验成功构建pCS-MEGp24核酸疫苗，证实了其可在真核细胞中表达目的基因，为其进一步深入研究和应用打下了基础。
[Abstract]:Acquired immunodeficiency syndrome (AIDS) is a malignant infectious disease caused by human jmmunodeficiency virus (HIV) infection. Since the disease was first discovered in the United States in 1981, there have been more than 65 million cases of infection worldwide, and AIDS is spreading at an alarming rate, posing a serious threat to human health. It is difficult for traditional vaccines to produce effective immune protection. Nucleic acid vaccine is a new immune control agent developed from gene therapy field. It can induce cellular immune response and humoral immune response at the same time. It is safe and flexible to construct and become a very promising candidate vaccine for AIDS. The pSFV1 vector is an eukaryotic expression vector derived from Semliki forest virus SFV RNA replicator of Semliki forest virus.However, when the vector system is used, it is necessary to transcribe the plasmid DNA into RNAs in vitro, which is very inconvenient to operate and limit its wide application. In this study, the replicon of SFV was inserted into the CMVie promoter of eukaryotic expression vector pIRESneo and the transcriptional termination signal of adenylate was inserted into the eukaryotic expression vector pIRESneo by molecular cloning technique. The DNA expression vector pCScontaining Semliki forest virus replicon was constructed. The modified vector could be prepared by routine DNA vaccine. The recombinant vector could be immunized, which overcame the drawback of in vitro transcription of the original vector pSFV1 for the preparation of RNA. The enhanced green fluorescent protein (EGFP) gene fragment was inserted into the polyclonal site of pCS vector and pCS-EGFP was transfected into BHK-21 cells. The enhanced green fluorescent protein (EGFP) was observed by fluorescence microscope. This new expression vector can be used to express foreign genes. In order to explore the possibility of using this novel vector pCS to develop antiviral vaccine, we selected the epitope of HIV dominant antigen and designed the antigen gene based on HIV-1 epitope gene. The HIV polyepitope gene, Multiple-epitope gene, was chimerized with HIV-1 giant particle p24. The HIV chimeric multiepitope MEGp24 gene was digested from the preserved pVAX1-MEGp24 enzyme and cloned into the pCS expression vector. The HIV polyepitope nucleic acid vaccine pCS-MEGp24was successfully constructed. Indirect immunofluorescence assay (IFA) showed that MEGp24 gene was effectively expressed in BHK-21 cells and the transcription product of MEGp24 gene was detected by RT-PCR. In conclusion, pCS-MEGp24 nucleic acid vaccine was successfully constructed, which confirmed that it could express the target gene in eukaryotic cells, which laid a foundation for its further research and application.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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