本文選題：HIV + 塞姆利基森林病毒復(fù)制子��； 參考：《延邊大學(xué)》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(chǎn)生有效的免疫保護。核酸疫苗是從基因治療領(lǐng)域發(fā)展起來的一種全新的免疫防治劑，可同時誘導(dǎo)細胞免疫應(yīng)答和體液免疫應(yīng)答，安全性好，構(gòu)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(fù)制子的真核表達載體，但此載體系統(tǒng)應(yīng)用時需在體外將質(zhì)粒DNA轉(zhuǎn)錄成RNA，操作很不方便，限制了其廣泛應(yīng)用。為此，本研究通過分子克隆技術(shù)，將SFV的復(fù)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(zhuǎn)錄終止信號Poly(A)之間，構(gòu)建了含塞姆利基森林病毒復(fù)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(zhuǎn)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(zhuǎn)染BHK-21細胞，熒光顯微鏡下能觀察到EGFP的表達產(chǎn)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(chǔ)，對抗原基因進行人工分子設(shè)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(gòu)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(gòu)建了含塞姆利基森林病毒復(fù)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(zhuǎn)染BHK-21細胞中得到有效表達，RT-PCR可檢測到MEGp24基因的轉(zhuǎn)錄產(chǎn)物mRNA。 綜上所述，本試驗成功構(gòu)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(yīng)用打下了基礎(chǔ)。
[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(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前10條

1 肖瑤,范秀娟,趙全壁,潘品良,戴傳斌,陳剛,王貴杰,邵一鳴;中國HIV-1B亞型P55和嵌合的P55-V3病毒樣顆粒候選疫苗免疫效果的研究[J];病毒學(xué)報;2000年04期

2 張健慧,Jens Wild,Kurt Bieler,Marcus Graf,Ludwig Deml,Hans Wolf,PeterLiljestrm,Ralf Wagner,邵一鳴;傳統(tǒng)DNA疫苗載體與Semliki森林病毒復(fù)制子對HIV-1 Pr55~(gag)表達與體液免疫原性的比較性研究[J];病毒學(xué)報;2002年01期

3 鄧瑤,孟昕,許洪林,王世峰,陸柔劍,王文玲,谷淑燕,阮力;Semliki森林病毒衍生的DNA疫苗與常規(guī)DNA疫苗的比較[J];病毒學(xué)報;2002年04期

4 許信剛,張彥明;“自殺性”DNA疫苗研究進展[J];動物醫(yī)學(xué)進展;2004年06期

5 李衛(wèi)華,趙連三;甲病毒表達載體的特點及在核酸疫苗中應(yīng)用[J];華西醫(yī)學(xué);2005年02期

6 李敬云;HIV的病原學(xué)研究進展[J];科技導(dǎo)報;2005年07期

7 程德春,郭俊杰,錢麗麗;HIV疫苗的研究進展[J];齊齊哈爾醫(yī)學(xué)院學(xué)報;2005年03期

8 羅玉子,仇華吉,劉建華,韓凌霞,李娜,張守發(fā),童光志;豬2型圓環(huán)病毒ORF2基因在塞姆利基森林病毒RNA復(fù)制子衍生的新型真核表達載體中的表達[J];生物工程學(xué)報;2004年06期

9 余云舟,孫志偉,俞煒源;基于DNA和RNA的雙功能Semliki森林病毒復(fù)制子載體的構(gòu)建[J];生物工程學(xué)報;2005年05期

10 李娜,仇華吉,李國新,朱慶虎,羅玉子,賈洪林,童光志;基于SemlikiForest病毒RNA復(fù)制子的豬瘟RNA疫苗的初步研究[J];中國生物工程雜志;2005年01期

相關(guān)博士學(xué)位論文 前1條

1 宋英今;HIV-1治療性重組DNA疫苗的構(gòu)建、純化及實驗免疫研究[D];吉林大學(xué);2005年

相關(guān)碩士學(xué)位論文 前1條

1 羅玉子;基于Semliki Forest病毒RNA復(fù)制子的表達載體及其衍生的2型豬圓環(huán)病毒RNA疫苗的初步研究[D];延邊大學(xué);2004年

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