本文選題：Red同源重組 + 黏附性大腸桿菌��； 參考：《沈陽藥科大學(xué)》2007年碩士論文

【摘要】： 疾病預(yù)防的有效途徑之一是接種疫苗，繼第一代減毒或無毒疫苗和第二代亞單位疫苗之后出現(xiàn)了第三代DNA疫苗。疫苗效果在很大程度上依賴于輸送抗原的載體。為研究更有效的疫苗載體形式，本研究應(yīng)用噬菌體Red重組酶介導(dǎo)的新型重組工程技術(shù)，構(gòu)建了多價DNA疫苗載體前體及攜帶禽流感病毒抗原基因的多價DNA疫苗載體，為了更好的輸送多價DNA疫苗，探索構(gòu)建了具有哺乳動物上皮細(xì)胞黏附能力的無毒性重組大腸桿菌活菌疫苗菌株。 在本室前期工作基礎(chǔ)上，本實(shí)驗(yàn)應(yīng)用如下Red重組工程系統(tǒng)及技術(shù)：①大腸桿菌染色體上Red重組系統(tǒng)及kan-sacB選擇反選擇篩選方法②pKD46重組系統(tǒng)及kan-kil選擇反選擇篩選方法③pYM-Red重組系統(tǒng)及Gap-Repair缺口修復(fù)技術(shù)，，進(jìn)行染色體上基因的無痕敲入、替換及質(zhì)粒構(gòu)建。本實(shí)驗(yàn)解決基因融合打靶問題時，在重組技術(shù)上有創(chuàng)新應(yīng)用，利用Red重組酶工作原理，將外源片段體外重疊PCR法制各改為片段體內(nèi)重組融合，片段融合及重組均在體內(nèi)完成，省略了體外PCR的步驟加快實(shí)驗(yàn)進(jìn)程。 以pcDNA3.1(-)B為基礎(chǔ)，以酶切連接方法構(gòu)建了四價DNA疫苗載體pWYⅠ-Ⅱ-Ⅲ-Ⅳ，瞬時轉(zhuǎn)染實(shí)驗(yàn)證明其具有蛋白表達(dá)能力，真核表達(dá)的多價DNA疫苗載體構(gòu)建成功，可用于攜帶多價抗原基因，避免了多質(zhì)粒操作帶來的不便。然后利用重組技術(shù)試圖將禽流感病毒基因組抗原基因HA頭部、NA全長分別加載到其Ⅰ、Ⅱ位，構(gòu)建二價DNA疫苗。 將南美錐蟲(trypanosome cruzi)gp82基因的P4及P8 DNA區(qū)段融合構(gòu)建成P4／8基因片段，將其敲入大腸桿菌染色體的外膜蛋白LamB基因內(nèi)形成融合蛋白，并展示在細(xì)胞外膜上，構(gòu)建出帶有細(xì)胞黏附能力的Ecoli DY330 P4／8lamb菌株。通過體外黏附實(shí)驗(yàn)證明，以前構(gòu)建的Ecoli DY330 P4lamb比新構(gòu)建的Ecoli DY330 P4／8lamb菌株黏附哺乳動物上皮細(xì)胞的能力要強(qiáng)；同時體外細(xì)胞毒性實(shí)驗(yàn)證明DY330 P4lamb菌株安全性良好，可做為活菌疫苗載體的候選株進(jìn)行深入研究。 研究表明，重組工程技術(shù)在構(gòu)建多種形式疫苗載體中具有靈活性及有效性，構(gòu)建的多價DNA疫苗載體具備通用性，可以加載多種抗原基因的組合，作為快速制備疫苗的模板，有效應(yīng)對突發(fā)疫情；構(gòu)建的黏附性活菌疫苗載體可以攜帶DNA疫苗，也可以直接在染色體上攜帶抗原基因，高效運(yùn)送抗原基因或蛋白。在應(yīng)用中不斷嘗試各種構(gòu)建方案也使我們更深入理解了重組酶工作原理，希望通過技術(shù)的拓展加速疫苗的研制與開發(fā)進(jìn)程，為疾病的防治作出貢獻(xiàn)。
[Abstract]:One of the effective methods of disease prevention is vaccination. After the first generation attenuated or nontoxic vaccine and the second generation subunit vaccine, the third generation DNA vaccine appeared. The efficacy of the vaccine depends largely on the carrier that carries the antigen. In order to study the more effective form of vaccine vector, a novel recombinant engineering technique mediated by phage Red recombinant enzyme was used to construct multivalent DNA vaccine vector precursor and multivalent DNA vaccine vector carrying avian influenza virus antigen gene. In order to better transport multivalent DNA vaccine, a nontoxic recombinant Escherichia coli vaccine strain with the ability of mammalian epithelial cell adhesion was constructed. On the basis of our earlier work, In this experiment, the following Red recombination engineering systems and techniques were used: the Red recombination system on the chromosome of Escherichia coli: 1, the kan-sacB selection reverse selection screening method, the 2pKD46 recombination system, the kan-kil selection reverse selection screening method, the 3pYM-Red recombination system, and the Gap-Repair notch repair technique. The genes on chromosome were knockin, substitution and plasmid construction. In order to solve the problem of gene fusion targeting, this experiment has innovative application in recombination technology. Using the principle of Red recombinant enzyme, the method of external fragment overlapping PCR in vitro is changed into fragment in vivo recombination, and the fragment fusion and recombination are completed in vivo. The steps of in vitro PCR were omitted to accelerate the experimental process. On the basis of pcDNA3.1(-)B, the tetravalent DNA vaccine vector pWY 鈪

本文編號：1946732