大肠杆菌偏嗜性人γ干扰素基因编码序列的克隆与表达

发布时间：2018-06-15 08:08

本文选题：人γ干扰素 + 密码子　；参考：《天津医科大学》2005年硕士论文

【摘要】：γ干扰素(Interferon-γ,IFN-γ)又称免疫干扰素(Immune interferon),是由淋巴细胞分泌的一类功能调节蛋白。除具有抗病毒、抗肿瘤的作用外,它还是人体内一种双向免疫调节因子,在治疗自身免疫性疾病方面有广阔的应用前景。天然人γ干扰素(hlFN-γ)来源有限、产量甚微,不能满足科研和临床应用的需要。因此,利用基因工程方法获得高表达的重组人γ干扰素(rhlFN-γ)成为国内外学者研究的热点。本文依据遗传密码具有简并性的特点,在不改变氨基酸顺序的条件下,用大肠杆菌基因序列中使用频率高的强密码子取代hlFN-γ基因编码序列中的弱密码子,并将修改后的编码序列分为六段,进行人工化学合成。利用PCR技术扩增出目的基因后,将其克隆到表达载体pBV220上,进而转化入E.coli JM109中进行诱导表达。经SDS-PAGE和Western Blotting检测证实,表达产物的分子量约为18KD,与预期相符。通过优化表达条件,可提高目的蛋白表达产量,使其占菌体总蛋白的39.9%。重组蛋白rhlFN-γ主要存在于E.coli JM109的包涵体中,纯化时将其自包涵体中溶出后,采用分步稀释的方法进行复性。梯度透析后获得的rhlFN-γ样品委托中国药品生物制品检定所对其抗病毒活性进行测定,其活性效价为5.6×10~3IU/ml,比活性为2.8×10~4IU/mg。
[Abstract]:Interferon 纬 Interferon- 纬 (IFN- 纬), also known as immune interferon, is a kind of functional regulatory protein secreted by lymphocytes. In addition to its antiviral and anti-tumor effects, it is also a bidirectional immunomodulatory factor in human body, and has a broad application prospect in the treatment of autoimmune diseases. The source of natural human interferon 纬 hlFN- 纬 is limited and the yield is very small, which can not meet the needs of scientific research and clinical application. Therefore, the high expression recombinant human interferon 纬 (rhlFN- 纬) obtained by genetic engineering has become a hot research topic at home and abroad. Based on the degeneracy of genetic code and without changing the sequence of amino acids, the weak codon of hlFN- 纬 gene was replaced by a strong codon with high frequency in Escherichia coli gene sequence. The modified coding sequence was divided into six segments for artificial chemical synthesis. The target gene was amplified by PCR and cloned into the expression vector pBV220, which was transformed into E. coli JM109 for induction expression. SDS-PAGE and Western blotting showed that the molecular weight of the expressed product was about 18kD, which was in accordance with the expectation. By optimizing the expression conditions, the expression yield of the target protein can be increased, which accounts for 39.9% of the total bacterial protein. The recombinant protein rhlFN- 纬 was mainly found in the inclusion body of E. coli JM109. After being purified from the inclusion body, the recombinant protein was renatured by a step dilution method. The rhlFN- 纬 sample obtained after gradient dialysis was entrusted to the Chinese Institute for the Control of Pharmaceutical and Biological products for the determination of its antiviral activity. The titer of its antiviral activity was 5.6 脳 10 ~ (-3) U / ml, and its specific activity was 2.8 脳 10 ~ (4) IUU / mg.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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