人Tum-5基因重组逆转录病毒载体系统的建立与分析

发布时间：2018-06-16 00:48

本文选题：Tum-5基因 + pLXSN　；参考：《北华大学》2008年硕士论文

【摘要】：目的:构建携带人肿瘤抑素基因(Tumstatin)抗血管生成活性片段Tum-5基因的重组逆转录病毒载体,使用包装细胞PA317将其包装成假病毒颗粒,并用NIH3T3细胞对其进行检测分析。方法:用Trizol试剂从胎肾组织中提取总RNA,用RT-PCR法扩增Tum-5片段,并将扩增片段定向克隆到逆转录病毒质粒pLXSN的MCS。将获得的重组质粒利用电穿孔方法转染PA317细胞,经G418筛选阳性克隆并扩增培养,收集其产生的含Tum-5的假病毒颗粒,用NIH3T3检测其病毒滴度及安全性。结果:用RT-PCR方法获得了255bp的Tum-5基因序列(含酶切位点),并将其成功的定向克隆到逆转录病毒质粒pLXSN,测序结果表明克隆出的基因序列与Gene Bank中所提供的Tum-5基因序列完全一致。RT-PCR证实,转染后的PA317、NIH3T3细胞中存在人Tum-5特异性片段,病毒滴度为2.05×104cfu/ml,并且没有对NIH3T3细胞造成二次感染。结论:成功的构建了携带人Tum-5基因的逆转录病毒载体系统,并对其性状及疗效进行了相关检测,为肿瘤的抗血管生成治疗以及血管依赖性疾病的基因治疗提供了一种全新的、有效的、安全的外源性基因导入的途径。
[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.
【学位授予单位】：北华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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