【摘要】： 第一部分畢赤酵母表達(dá)可溶性hsBAFF的純化及生物學(xué)性質(zhì)研究 B淋巴細(xì)胞刺激因子(BhFF)是1999年發(fā)現(xiàn)的腫瘤壞死因子超家族成員。BAFF在anti-IgM存在下能專一的刺激B細(xì)胞增殖和分化，因而在體液免疫中有重要的作用。其缺陷或過量表達(dá)均能引起機(jī)體的免疫失衡，從而誘發(fā)多種疾病，因此，BAFF以及抑制劑的開發(fā)有望成為治療人體免疫缺陷和自身免疫性疾病的新型藥物。鑒于此，我們利用常規(guī)的PCR克隆方法，將hsBAFF的cDNA克隆至畢赤酵母表達(dá)載體pPIC9中，SDS-PAGE和Western blotting分析表明所分泌蛋白為糖基化的hsBAFF。重組蛋白經(jīng)離子交換和分子篩層析純度為95％，得率為1升發(fā)酵液可得102毫克hsBAFF，氨端測(cè)序后再次表明蛋白為hsBAFF。結(jié)果顯示畢赤酵母表達(dá)系統(tǒng)表達(dá)hsBAFF可以滿足科研甚至工業(yè)化生產(chǎn)目的。 第二部分昆蟲桿狀病毒靶向于肝癌基因治療的研究 現(xiàn)已發(fā)現(xiàn)桿狀病毒可進(jìn)入某些哺乳動(dòng)物細(xì)胞，并對(duì)肝細(xì)胞具有一定的靶向性，這提示可將桿狀病毒作為一種對(duì)肝癌基因治療的新型載體。本文對(duì)桿狀病毒載體的改造及對(duì)肝癌細(xì)胞的靶向凋亡進(jìn)行了進(jìn)一步的研究。以綠色熒光蛋白基因?yàn)閳?bào)告基因，利用Bac-to-Bac系統(tǒng)構(gòu)建了分別含有CMV和hTERT啟動(dòng)子表達(dá)盒的兩種重組桿狀病毒�？捎^察到CMV啟動(dòng)子在Sf9細(xì)胞中可啟動(dòng)報(bào)告基因的表達(dá)，，兩種病毒在相同的感染復(fù)數(shù)下對(duì)HepG2和Hela細(xì)胞具有不同的表達(dá)效率。我們又將凋亡素基因置于hTERT啟動(dòng)子下，構(gòu)建的重組桿狀病毒BacV-hTERT-Apoptin誘導(dǎo)了HepG2細(xì)胞的凋亡。因此可以認(rèn)為，重組桿狀病毒BacV-hTERT-Apoptin可作為一種對(duì)肝癌靶向基因治療的簡便高效的基因治療載體。
[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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R735.7;R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 全碩,潘國宗,魯重美,劉東遠(yuǎn),左瑾,方福德;甲胎蛋白特異啟動(dòng)元件介導(dǎo)的肝癌靶向性基因治療的體外實(shí)驗(yàn)研究[J];中國醫(yī)學(xué)科學(xué)院學(xué)報(bào);1999年02期

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