本文選題：Tum-5基因 + pLXSN�。� 參考：《北華大學》2008年碩士論文

【摘要】：目的:構(gòu)建攜帶人腫瘤抑素基因(Tumstatin)抗血管生成活性片段Tum-5基因的重組逆轉(zhuǎn)錄病毒載體,使用包裝細胞PA317將其包裝成假病毒顆粒,并用NIH3T3細胞對其進行檢測分析。 方法:用Trizol試劑從胎腎組織中提取總RNA,用RT-PCR法擴增Tum-5片段,并將擴增片段定向克隆到逆轉(zhuǎn)錄病毒質(zhì)粒pLXSN的MCS。將獲得的重組質(zhì)粒利用電穿孔方法轉(zhuǎn)染PA317細胞,經(jīng)G418篩選陽性克隆并擴增培養(yǎng),收集其產(chǎn)生的含Tum-5的假病毒顆粒,用NIH3T3檢測其病毒滴度及安全性。 結(jié)果:用RT-PCR方法獲得了255bp的Tum-5基因序列(含酶切位點),并將其成功的定向克隆到逆轉(zhuǎn)錄病毒質(zhì)粒pLXSN,測序結(jié)果表明克隆出的基因序列與Gene Bank中所提供的Tum-5基因序列完全一致。RT-PCR證實,轉(zhuǎn)染后的PA317、NIH3T3細胞中存在人Tum-5特異性片段,病毒滴度為2.05×104cfu/ml,并且沒有對NIH3T3細胞造成二次感染。 結(jié)論:成功的構(gòu)建了攜帶人Tum-5基因的逆轉(zhuǎn)錄病毒載體系統(tǒng),并對其性狀及療效進行了相關(guān)檢測,為腫瘤的抗血管生成治療以及血管依賴性疾病的基因治療提供了一種全新的、有效的、安全的外源性基因?qū)氲耐緩健?br/>[Abstract]:Aim: to construct a recombinant retroviral vector carrying the antiangiogenic activity fragment Tum-5 of human tumor endostatin gene (Tumstatin). The recombinant retroviral vector was packaged into pseudoviral particles by packaging cell line PA317 and detected by NIH3T3 cells. Methods: total RNAs were extracted from fetal kidney tissue with Trizol reagent. Tum-5 fragment was amplified by RT-PCR and cloned into MCSs of retrovirus plasmid pLXSN. The obtained recombinant plasmid was transfected into PA317 cells by electroporation. The positive clones were screened by G418 and cultured. The pseudoviral particles containing Tum-5 were collected and the virus titer and safety of PA317 cells were detected by NIH3T3. Results: the Tum-5 gene sequence of 255bp was obtained by RT-PCR, and successfully cloned into retrovirus plasmid pLXSN.The sequencing results showed that the cloned gene sequence was identical with the Tum-5 gene sequence provided by Gene Bank. Completely consistent. RT-PCR confirmed that, There was a human Tum-5 specific fragment in the transfected PA317- NIH3T3 cells, the titer of the virus was 2.05 脳 104 cfur / ml, and no secondary infection was caused to NIH3T3 cells. Conclusion: the retroviral vector system carrying human Tum-5 gene was successfully constructed, and its character and curative effect were detected, which provided a new method for anti-angiogenesis therapy and gene therapy of vascular dependent diseases. An effective, safe approach to exogenous gene transfer.
【學位授予單位】：北華大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346

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