含绿色荧光蛋白基因的优化新型HIV-1慢病毒载体的构建及表达
发布时间：2019-05-15 23:49
【摘要】： HIV-1慢病毒载体可感染非分裂细胞,具有转移外源基因片段大、使目的基因长期稳定表达、不易诱发宿主免疫反应等优点,是脑梗死基因治疗的理想载体。本实验在慢病毒载体三质粒包装系统基础上,将野生型HIV-1基因组分装在四个质粒中,构建优化新型HIV-1慢病毒载体四质粒包装系统:载体质粒(含指示基因——绿色荧光蛋白基因)、gag-pol质粒(包装质粒)、rev质粒和包膜质粒,经限制性酶切、电泳,证实质粒构建成功。然后经转化感受态细胞、摇菌,大量提取质粒,以备转染和测定载体滴度使用。 将四质粒包装系统共转染293T包装细胞,48小时后荧光显微镜观察可见绿色荧光,证实质粒转入细胞。经收集细胞上清及高速离心获得HIV-1慢病毒载体颗粒。病毒载体再次转染293T细胞,仍可观察到绿色荧光,说明载体携带的指示基因成功融合入细胞基因组。经测定病毒滴度为4×108TU/ml,这说明假包膜VSV-G使载体颗粒的稳定性提高,通过高速离心可获得较高滴度的病毒颗粒。优化新型HIV-1慢病毒载体构建成功。四质粒系统的使用进一步减少了各质粒间的同源性,减少了重组产生有复制能力病毒的机会。
[Abstract]:HIV-1 lentivirus vector can infect non-mitotic cells and has the advantages of large transfer of foreign gene fragments, stable expression of the target gene for a long time, and is not easy to induce host immune response. It is an ideal vector for gene therapy of cerebral infarction. In this experiment, the wild type HIV-1 genome was divided into four plasmids on the basis of lentivirus vector three plasmid packaging system. A novel HIV-1 lentivirus vector four-plasmid packaging system was constructed: vector plasmid (containing indicator gene-green fluorescent protein gene), gag-pol plasmid (packaging plasmid), rev plasmid and envelope plasmid, restriction enzyme digestion, electrophoresis, It was confirmed that the plasmid was constructed successfully. After transformation of receptive cells, shaking bacteria, a large number of plasmid was extracted for transfection and determination of vector titer. The four plasmid packaging system was co-transfected into 293T packaging cells. 48 hours later, green fluorescence was observed by fluorescence microscope, which confirmed that the plasmid was transferred into the cells. HIV-1 lentivirus vector particles were obtained by collecting cell culture and high speed centrifugation. Green fluorescence could still be observed when 293T cells were re-transfected with virus vector, which indicated that the indicator gene carried by the vector was successfully integrated into the cell genome. The titer of the virus was 4 脳 10 ~ 8TU / ml, which indicated that the stability of the carrier particles was improved by pseudocapsule VSV-G, and the virus particles with high titer could be obtained by high speed centrifugation. The construction of a novel HIV-1 lentivirus vector was optimized successfully. The use of the four plasmid system further reduced the homology between the plasmids and reduced the chance of recombination to produce replicative viruses.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
本文编号：2477857
[Abstract]:HIV-1 lentivirus vector can infect non-mitotic cells and has the advantages of large transfer of foreign gene fragments, stable expression of the target gene for a long time, and is not easy to induce host immune response. It is an ideal vector for gene therapy of cerebral infarction. In this experiment, the wild type HIV-1 genome was divided into four plasmids on the basis of lentivirus vector three plasmid packaging system. A novel HIV-1 lentivirus vector four-plasmid packaging system was constructed: vector plasmid (containing indicator gene-green fluorescent protein gene), gag-pol plasmid (packaging plasmid), rev plasmid and envelope plasmid, restriction enzyme digestion, electrophoresis, It was confirmed that the plasmid was constructed successfully. After transformation of receptive cells, shaking bacteria, a large number of plasmid was extracted for transfection and determination of vector titer. The four plasmid packaging system was co-transfected into 293T packaging cells. 48 hours later, green fluorescence was observed by fluorescence microscope, which confirmed that the plasmid was transferred into the cells. HIV-1 lentivirus vector particles were obtained by collecting cell culture and high speed centrifugation. Green fluorescence could still be observed when 293T cells were re-transfected with virus vector, which indicated that the indicator gene carried by the vector was successfully integrated into the cell genome. The titer of the virus was 4 脳 10 ~ 8TU / ml, which indicated that the stability of the carrier particles was improved by pseudocapsule VSV-G, and the virus particles with high titer could be obtained by high speed centrifugation. The construction of a novel HIV-1 lentivirus vector was optimized successfully. The use of the four plasmid system further reduced the homology between the plasmids and reduced the chance of recombination to produce replicative viruses.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
【参考文献】
相关期刊论文 前4条
1 黄东煜;张志坚;陈柏龄;吴秀丽;王柠;张彦定;;大鼠Bdnf基因慢病毒载体的构建及其在骨髓间质干细胞中的表达[J];生物工程学报;2007年02期
2 陈柏龄;张志坚;黄东煜;吴秀丽;张彦定;;血管生长素-1基因慢病毒载体的构建及其在间质干细胞的表达[J];中国病理生理杂志;2007年08期
3 万虹;刘松;陈刚;历俊华;;HIV-1来源的慢病毒载体转染大鼠脊髓神经干细胞[J];中华神经外科杂志;2006年12期
4 李振宇,徐开林,潘秀英,鹿群先,何徐彭,孙海英,主鸿鹄;HIV-1慢病毒载体的构建及结构改造[J];中华血液学杂志;2004年09期
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