Ⅰ.毕赤酵母表达可溶性hsBAFF的纯化及生物学性质研究 Ⅱ.昆虫杆状病毒靶向肝癌基因治疗的研究
发布时间：2018-11-23 17:31
【摘要】： 第一部分毕赤酵母表达可溶性hsBAFF的纯化及生物学性质研究 B淋巴细胞刺激因子(BhFF)是1999年发现的肿瘤坏死因子超家族成员。BAFF在anti-IgM存在下能专一的刺激B细胞增殖和分化，因而在体液免疫中有重要的作用。其缺陷或过量表达均能引起机体的免疫失衡，从而诱发多种疾病，因此，BAFF以及抑制剂的开发有望成为治疗人体免疫缺陷和自身免疫性疾病的新型药物。鉴于此，我们利用常规的PCR克隆方法，将hsBAFF的cDNA克隆至毕赤酵母表达载体pPIC9中，SDS-PAGE和Western blotting分析表明所分泌蛋白为糖基化的hsBAFF。重组蛋白经离子交换和分子筛层析纯度为95％，得率为1升发酵液可得102毫克hsBAFF，氨端测序后再次表明蛋白为hsBAFF。结果显示毕赤酵母表达系统表达hsBAFF可以满足科研甚至工业化生产目的。 第二部分昆虫杆状病毒靶向于肝癌基因治疗的研究 现已发现杆状病毒可进入某些哺乳动物细胞，并对肝细胞具有一定的靶向性，这提示可将杆状病毒作为一种对肝癌基因治疗的新型载体。本文对杆状病毒载体的改造及对肝癌细胞的靶向凋亡进行了进一步的研究。以绿色荧光蛋白基因为报告基因，利用Bac-to-Bac系统构建了分别含有CMV和hTERT启动子表达盒的两种重组杆状病毒。可观察到CMV启动子在Sf9细胞中可启动报告基因的表达，，两种病毒在相同的感染复数下对HepG2和Hela细胞具有不同的表达效率。我们又将凋亡素基因置于hTERT启动子下，构建的重组杆状病毒BacV-hTERT-Apoptin诱导了HepG2细胞的凋亡。因此可以认为，重组杆状病毒BacV-hTERT-Apoptin可作为一种对肝癌靶向基因治疗的简便高效的基因治疗载体。
[Abstract]:Purification and Biological Properties of soluble hsBAFF expressed by Pichia pastoris; B lymphocyte stimulating factor (BhFF) is a member of tumor necrosis factor superfamily discovered in 1999. BAFF in anti-IgM B cell proliferation and differentiation can be specifically stimulated in the presence of, Therefore, it plays an important role in humoral immunity. Its deficiency or overexpression can cause immune imbalance and induce many diseases. Therefore, the development of BAFF and inhibitors is expected to be a new drug for the treatment of human immune deficiency and autoimmune diseases. In view of this, the cDNA of hsBAFF was cloned into Pichia pastoris expression vector pPIC9 by conventional PCR cloning method. SDS-PAGE and Western blotting analysis showed that the secreted protein was glycosylated hsBAFF.. The purity of recombinant protein was 95% by ion exchange and molecular sieve chromatography, and the yield was 1 litre fermentation broth, 102mg hsBAFF, was sequenced and the protein was hsBAFF. again. The results showed that the expression of hsBAFF by Pichia pastoris expression system could meet the purpose of scientific research and even industrial production. The second part of the study of insect baculovirus targeting liver cancer gene therapy has found that baculovirus can enter some mammalian cells and has a certain targeting to liver cells. This suggests that baculovirus can be used as a novel vector for gene therapy of liver cancer. In this paper, the modification of baculovirus vector and the targeting apoptosis of hepatoma cells were further studied. Using green fluorescent protein as reporter gene, two recombinant baculoviruses containing CMV and hTERT promoter boxes were constructed by using Bac-to-Bac system. It was observed that CMV promoter could initiate the expression of reporter gene in Sf9 cells. The two viruses had different expression efficiency on HepG2 and Hela cells under the same infection complex number. The apoptin gene was placed in the hTERT promoter and the recombinant baculovirus BacV-hTERT-Apoptin induced the apoptosis of HepG2 cells. It is suggested that recombinant baculovirus BacV-hTERT-Apoptin can be used as a simple and efficient gene therapy vector for liver cancer targeting gene therapy.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R735.7;R346
本文编号：2352241
[Abstract]:Purification and Biological Properties of soluble hsBAFF expressed by Pichia pastoris; B lymphocyte stimulating factor (BhFF) is a member of tumor necrosis factor superfamily discovered in 1999. BAFF in anti-IgM B cell proliferation and differentiation can be specifically stimulated in the presence of, Therefore, it plays an important role in humoral immunity. Its deficiency or overexpression can cause immune imbalance and induce many diseases. Therefore, the development of BAFF and inhibitors is expected to be a new drug for the treatment of human immune deficiency and autoimmune diseases. In view of this, the cDNA of hsBAFF was cloned into Pichia pastoris expression vector pPIC9 by conventional PCR cloning method. SDS-PAGE and Western blotting analysis showed that the secreted protein was glycosylated hsBAFF.. The purity of recombinant protein was 95% by ion exchange and molecular sieve chromatography, and the yield was 1 litre fermentation broth, 102mg hsBAFF, was sequenced and the protein was hsBAFF. again. The results showed that the expression of hsBAFF by Pichia pastoris expression system could meet the purpose of scientific research and even industrial production. The second part of the study of insect baculovirus targeting liver cancer gene therapy has found that baculovirus can enter some mammalian cells and has a certain targeting to liver cells. This suggests that baculovirus can be used as a novel vector for gene therapy of liver cancer. In this paper, the modification of baculovirus vector and the targeting apoptosis of hepatoma cells were further studied. Using green fluorescent protein as reporter gene, two recombinant baculoviruses containing CMV and hTERT promoter boxes were constructed by using Bac-to-Bac system. It was observed that CMV promoter could initiate the expression of reporter gene in Sf9 cells. The two viruses had different expression efficiency on HepG2 and Hela cells under the same infection complex number. The apoptin gene was placed in the hTERT promoter and the recombinant baculovirus BacV-hTERT-Apoptin induced the apoptosis of HepG2 cells. It is suggested that recombinant baculovirus BacV-hTERT-Apoptin can be used as a simple and efficient gene therapy vector for liver cancer targeting gene therapy.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R735.7;R346
【参考文献】
相关期刊论文 前1条
1 全硕,潘国宗,鲁重美,刘东远,左瑾,方福德;甲胎蛋白特异启动元件介导的肝癌靶向性基因治疗的体外实验研究[J];中国医学科学院学报;1999年02期
本文编号：2352241
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2352241.html
最近更新
教材专著
