本文選題：HIV + 塞姆利基森林病毒復制子�。� 參考：《延邊大學》2007年碩士論文

【摘要】： 獲得性免疫缺陷綜合征(Acquired immunodeficiency syndrome，AIDS)即艾滋病，是由人免疫缺陷病毒(Human jmmunodeficiency Virus，HIV)感染引起的一種惡性傳染病。自從1981年在美國首次發(fā)現(xiàn)該病以來，全球已有6 500多萬感染病例，艾滋病還在以驚人的速度蔓延著，嚴重威脅人類的健康。HIV基因高度變異，逃避免疫監(jiān)視，使得傳統(tǒng)疫苗較難產生有效的免疫保護。核酸疫苗是從基因治療領域發(fā)展起來的一種全新的免疫防治劑，可同時誘導細胞免疫應答和體液免疫應答，安全性好，構建靈活，成為一種非常有希望的艾滋病候選疫苗。 pSFV1載體為衍生于塞姆利基森林病毒(Semliki forest virus，SFV)RNA復制子的真核表達載體，但此載體系統(tǒng)應用時需在體外將質粒DNA轉錄成RNA，操作很不方便，限制了其廣泛應用。為此，本研究通過分子克隆技術，將SFV的復制子插入到真核表達載體pIRESneo的CMVie立即早期啟動子和腺苷酸轉錄終止信號Poly(A)之間，構建了含塞姆利基森林病毒復制子的DNA表達載體pCS，改造后的載體可按常規(guī)DNA疫苗的方式進行制備、免疫，克服了原載體pSFV1體外轉錄制備RNA的弊端。將增強型綠色熒光蛋白(EGFP)基因片段插入到pCS載體多克隆位點中，得到pCS-EGFP，用其轉染BHK-21細胞，熒光顯微鏡下能觀察到EGFP的表達產物增強型綠色熒光蛋白，表明該新型表達載體可以用于表達外源基因。 為了探討此新型載體pCS用于研制抗病毒疫苗的可能性，本研究選擇HIV優(yōu)勢抗原表位，以HIV-1表位基因為基礎，對抗原基因進行人工分子設計，將HIV多表位基因(Multiple-epitope gene，，MEG)與HIV-1巨分子顆粒p24進行嵌合。從已構建保存的pVAX1-MEGp24酶切下HIV嵌合多表位MEGp24基因，克隆到pCS表達載體中，成功構建了含塞姆利基森林病毒復制子的HIV多表位核酸疫苗pCS-MEGp24。間接免疫熒光試驗(IFA)檢測表明MEGp24基因在轉染BHK-21細胞中得到有效表達，RT-PCR可檢測到MEGp24基因的轉錄產物mRNA。 綜上所述，本試驗成功構建pCS-MEGp24核酸疫苗，證實了其可在真核細胞中表達目的基因，為其進一步深入研究和應用打下了基礎。
[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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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