特異性艾滋病核酸疫苗轉(zhuǎn)導(dǎo)載體的構(gòu)建及初步應(yīng)用
發(fā)布時間：2018-07-28 07:31
【摘要】： 當下,在全世界對控制和預(yù)防艾滋病病毒(Human immunodeficiency virus,HIV)感染工作中尚無完全有效手段的前提下,要有效的預(yù)防并控制艾滋病病毒的感染,最佳的思路是開發(fā)出一種能夠安全、有效的預(yù)防艾滋病病毒感染的疫苗。 本研究的目的是構(gòu)建一種新型的具有特異性的基因轉(zhuǎn)導(dǎo)載體TG蛋白�；诖┠る男盘栯�(TAT)、DNA識別及結(jié)合序列系統(tǒng)(GAL4/UAS)上,添加適當?shù)募兓拌b定標簽His-tag,定向克隆得到編碼TG蛋白的基因片段。以原核表達系統(tǒng)中的pET-28a為載體構(gòu)建了并得到了原核表達重組質(zhì)粒pET-TG。GAL4蛋白具有能夠與UAS核苷酸序列特異性結(jié)合的能力,所以經(jīng)過純化的TG蛋白在一定條件下可在機體外與嵌入熒光蛋白基因EGFP片段的pVAX1-UAS-EGFP質(zhì)粒特異性結(jié)合,然后再利用瓊脂糖凝膠電泳實驗就可以直觀的證明TG蛋白可與pVAX1-UAS-EGFP質(zhì)粒特異性結(jié)合。再摸索出TG蛋白與pVAX1-UAS-EGFP質(zhì)粒結(jié)合活性最高時的質(zhì)粒蛋白濃度和時間條件。在細胞水平上將蛋白與質(zhì)粒特異性的結(jié)合物產(chǎn)物與293T細胞共浴,最后在熒光顯微鏡下通過對熒光的檢測研究TG蛋白的細胞轉(zhuǎn)導(dǎo)活性。 通過大腸桿菌表達系統(tǒng)表達分子量為21kDa的目的蛋白。通過瓊脂糖凝膠電泳可以看出質(zhì)粒與蛋白結(jié)合活性最強時質(zhì)粒量為10μg,蛋白量為16μg,同時TG蛋白在10min內(nèi)即可與pVAX1-UAS-EGFP結(jié)合,2h內(nèi)其結(jié)合效率無明顯變化。通過細胞轉(zhuǎn)導(dǎo)發(fā)現(xiàn)pVAX1-UAS-EGFP質(zhì)粒與TG蛋白結(jié)合的結(jié)合物轉(zhuǎn)導(dǎo)293T細胞均出現(xiàn)特異性熒光,表明TAT基因具有穿膜作用的。本研究為艾滋病核酸疫苗轉(zhuǎn)導(dǎo)載體研究的進一步深入提供依據(jù)。
[Abstract]:At present, under the premise that there is no completely effective means to prevent and control HIV infection in the world, the best way to prevent and control HIV infection is to develop a safe one. Effective vaccine against HIV infection. The aim of this study was to construct a novel gene transduction vector TG protein. Based on the transmembrane peptide signal peptide (TAT) DNA recognition and binding sequence system (GAL4/UAS), the appropriate purification and identification tag His-tagwas added, and the gene fragment encoding TG protein was cloned. Using pET-28a in prokaryotic expression system as a vector, the prokaryotic expression plasmid pET-TG.GAL4 protein has the ability to bind specifically to UAS nucleotide sequence. Therefore, the purified TG protein can specifically bind to the pVAX1-UAS-EGFP plasmid embedded in the EGFP fragment of the fluorescent protein gene outside the body under certain conditions. Then agarose gel electrophoresis was used to demonstrate that TG protein could bind specifically to pVAX1-UAS-EGFP plasmid. The concentration and time of TG protein binding to pVAX1-UAS-EGFP plasmid were found out. The protein and plasmide-specific conjugate product were combined with 293T cells at the cellular level. Finally, the cell transduction activity of TG protein was studied by fluorescence microscopy. The target protein with molecular weight of 21kDa was expressed by E. coli expression system. Agarose gel electrophoresis showed that the amount of plasmid was 10 渭 g and the amount of protein was 16 渭 g when the binding activity between plasmid and protein was the highest, and the binding efficiency of TG protein to pVAX1-UAS-EGFP could not change significantly within 2 hours after binding to pVAX1-UAS-EGFP in 10min. Specific fluorescence was found in 293T cells transduced by pVAX1-UAS-EGFP plasmid binding to TG protein, indicating that TAT gene has transmembrane effect. This study provides the basis for the further study of AIDS nucleic acid vaccine transduction vector.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392.1
本文編號：2149362
[Abstract]:At present, under the premise that there is no completely effective means to prevent and control HIV infection in the world, the best way to prevent and control HIV infection is to develop a safe one. Effective vaccine against HIV infection. The aim of this study was to construct a novel gene transduction vector TG protein. Based on the transmembrane peptide signal peptide (TAT) DNA recognition and binding sequence system (GAL4/UAS), the appropriate purification and identification tag His-tagwas added, and the gene fragment encoding TG protein was cloned. Using pET-28a in prokaryotic expression system as a vector, the prokaryotic expression plasmid pET-TG.GAL4 protein has the ability to bind specifically to UAS nucleotide sequence. Therefore, the purified TG protein can specifically bind to the pVAX1-UAS-EGFP plasmid embedded in the EGFP fragment of the fluorescent protein gene outside the body under certain conditions. Then agarose gel electrophoresis was used to demonstrate that TG protein could bind specifically to pVAX1-UAS-EGFP plasmid. The concentration and time of TG protein binding to pVAX1-UAS-EGFP plasmid were found out. The protein and plasmide-specific conjugate product were combined with 293T cells at the cellular level. Finally, the cell transduction activity of TG protein was studied by fluorescence microscopy. The target protein with molecular weight of 21kDa was expressed by E. coli expression system. Agarose gel electrophoresis showed that the amount of plasmid was 10 渭 g and the amount of protein was 16 渭 g when the binding activity between plasmid and protein was the highest, and the binding efficiency of TG protein to pVAX1-UAS-EGFP could not change significantly within 2 hours after binding to pVAX1-UAS-EGFP in 10min. Specific fluorescence was found in 293T cells transduced by pVAX1-UAS-EGFP plasmid binding to TG protein, indicating that TAT gene has transmembrane effect. This study provides the basis for the further study of AIDS nucleic acid vaccine transduction vector.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392.1
【參考文獻】
相關(guān)期刊論文 前3條
1 王全楚,馮志華,聶青和,周永興;DC-SIGN: binding receptors for hepatitis C virus[J];Chinese Medical Journal;2004年09期
2 李海英;吳昊;;HIV致病機制研究進展[J];中國病原生物學雜志;2008年03期
3 ;Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency[J];World Journal of Gastroenterology;2007年44期
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