XAGE-1b腺病毒干擾載體的構(gòu)建及其功能研究與一種fiber缺失型重組腺病毒系統(tǒng)的構(gòu)建
發(fā)布時間：2018-11-20 07:06
【摘要】： XAGE是通過生物信息學(xué)方法發(fā)現(xiàn)的新一類腫瘤-睪丸抗原(cancer/testisantigen,CT抗原)基因,其主要表達(dá)亞型為XAGE-1b,其功能及表達(dá)調(diào)控機(jī)制的研究剛剛起步,已有的研究表明該基因可能與腫瘤細(xì)胞的生長和轉(zhuǎn)移相關(guān).在本研究的第一部分中,構(gòu)建了針對XAGE-1b進(jìn)行RNA干擾的重組腺病毒載體Adv-Sh1和Adv-Sh2.半定量和定量RT-PCR顯示Adv-Sh1比Adv-Sh2對XAGE-1b的干擾效率更高,細(xì)胞熒光干擾實(shí)驗(yàn)顯示Adv-Sh1對XAGE-1b有顯著的干擾作用.在細(xì)胞增殖和集落形成實(shí)驗(yàn)中,感染Adv-Sh1后的細(xì)胞增殖能力顯著降低(P＜0.01,Student's t檢驗(yàn)),形成的單克隆集落數(shù)也顯著減少.研究結(jié)果顯示:下調(diào)XAGE-1b基因表達(dá)對多種腫瘤細(xì)胞的增殖及轉(zhuǎn)移能力均有一定的降低作用,提示XAGE-1b在腫瘤生長和轉(zhuǎn)移過程中可能發(fā)揮重要作用.本研究為進(jìn)一步深入研究XAGE-1b的功能和致癌機(jī)制打下了良好基礎(chǔ). 腺病毒無法靶向感染某種特定的細(xì)胞,這主要是由于其纖毛蛋白(fiber)受體廣泛存在于各種細(xì)胞表面,因此限制了腺病毒作為基因治療載體的應(yīng)用.敲除腺病毒基因組中的fiber基因,替換為改造過的fiber基因是實(shí)現(xiàn)腺病毒靶向性的思路之一.在本研究的第二部分中,按照上述策略建立了一套新型的腺病毒靶向基因治療系統(tǒng).基于AdEasy腺病毒系統(tǒng)構(gòu)建了敲除fiber基因的腺病毒載體,同時構(gòu)建了穩(wěn)定表達(dá)Ad5 fiber基因的HEK293細(xì)胞系作為該病毒載體的包裝細(xì)胞系.結(jié)果表明該套系統(tǒng)產(chǎn)生的缺陷型病毒能夠在該細(xì)胞系中完成包裝并擴(kuò)增,具有正常病毒的感染能力.該系統(tǒng)為進(jìn)一步構(gòu)建靶向腺病毒基因治療系統(tǒng)打下了基礎(chǔ).
[Abstract]:XAGE is a new class of tumor-testicular antigen (cancer/testisantigen,CT) gene discovered by bioinformatics. The main expression subtype of XAGE-1b, is the function and regulation mechanism of XAGE-1b,. Previous studies have shown that the gene may be related to the growth and metastasis of tumor cells. In the first part of this study, we constructed recombinant adenovirus vectors Adv-Sh1 and Adv-Sh2. for RNA interference against XAGE-1b. Semi-quantitative and quantitative RT-PCR showed that Adv-Sh1 had higher interference efficiency to XAGE-1b than Adv-Sh2, and the cell fluorescence interference assay showed that Adv-Sh1 had a significant interference effect on XAGE-1b. In the experiment of cell proliferation and colony formation, the ability of cell proliferation was significantly decreased after Adv-Sh1 infection (P < 0.01), and the number of monoclonal colonies was also significantly decreased. The results showed that down-regulation of XAGE-1b gene expression could decrease the proliferation and metastasis of various tumor cells, suggesting that XAGE-1b might play an important role in tumor growth and metastasis. This study laid a good foundation for further study on the function and carcinogenic mechanism of XAGE-1b. Adenovirus can not be targeted to infect a specific cell, which is mainly due to the widespread presence of its ciliated protein (fiber) receptor on various cell surfaces, which limits the application of adenovirus as a gene therapy vector. Knockout of fiber gene in adenovirus genome and replacement with modified fiber gene is one of the ways to realize adenovirus targeting. In the second part of this study, a novel adenovirus targeting gene therapy system was established according to the above strategies. Adenovirus vector with fiber knockout was constructed based on AdEasy adenovirus system, and the HEK293 cell line stably expressing Ad5 fiber gene was constructed as the packaging cell line of the vector. The results showed that the defective virus produced by the system could be packaged and amplified in the cell line and had the ability to infect the normal virus. The system lays a foundation for further construction of target adenovirus gene therapy system.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373
本文編號：2344184
[Abstract]:XAGE is a new class of tumor-testicular antigen (cancer/testisantigen,CT) gene discovered by bioinformatics. The main expression subtype of XAGE-1b, is the function and regulation mechanism of XAGE-1b,. Previous studies have shown that the gene may be related to the growth and metastasis of tumor cells. In the first part of this study, we constructed recombinant adenovirus vectors Adv-Sh1 and Adv-Sh2. for RNA interference against XAGE-1b. Semi-quantitative and quantitative RT-PCR showed that Adv-Sh1 had higher interference efficiency to XAGE-1b than Adv-Sh2, and the cell fluorescence interference assay showed that Adv-Sh1 had a significant interference effect on XAGE-1b. In the experiment of cell proliferation and colony formation, the ability of cell proliferation was significantly decreased after Adv-Sh1 infection (P < 0.01), and the number of monoclonal colonies was also significantly decreased. The results showed that down-regulation of XAGE-1b gene expression could decrease the proliferation and metastasis of various tumor cells, suggesting that XAGE-1b might play an important role in tumor growth and metastasis. This study laid a good foundation for further study on the function and carcinogenic mechanism of XAGE-1b. Adenovirus can not be targeted to infect a specific cell, which is mainly due to the widespread presence of its ciliated protein (fiber) receptor on various cell surfaces, which limits the application of adenovirus as a gene therapy vector. Knockout of fiber gene in adenovirus genome and replacement with modified fiber gene is one of the ways to realize adenovirus targeting. In the second part of this study, a novel adenovirus targeting gene therapy system was established according to the above strategies. Adenovirus vector with fiber knockout was constructed based on AdEasy adenovirus system, and the HEK293 cell line stably expressing Ad5 fiber gene was constructed as the packaging cell line of the vector. The results showed that the defective virus produced by the system could be packaged and amplified in the cell line and had the ability to infect the normal virus. The system lays a foundation for further construction of target adenovirus gene therapy system.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 宋玉亮;彭思揚(yáng);王晨滔;朱乃碩;;XAGE-1b腺病毒表達(dá)載體的構(gòu)建及其功能研究[J];復(fù)旦學(xué)報(自然科學(xué)版);2008年03期
，本文編號：2344184
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