【作者】 王远鹏；

【导师】 龚兴国；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 人胸腺素α1 (Tα1)是一种28个氨基酸组成的具有免疫增强作用的生物活性多肽,其具有广泛的生物学功能,如免疫调节、抗病毒、抗肿瘤、内分泌调节等。目前Tal被广泛应用于慢性病毒性肝炎、细菌或真菌感染、免疫缺陷以及肿瘤等疾病的治疗,具有重大的经济效益与社会价值,因此Tα1的功能研究与生产制备的探索都具有重要意义。本文将Tα1基因转入大肠杆菌中表达并进行化学修饰,同时研究其对肿瘤细胞的周期阻滞作用。本实验首先以实验室保存的核酸片段为模板,人工合成了Tα1的核酸序列,将其与pTWINⅠ质粒中的内含肽自剪切序列(Ssp)相连,通过EcoRⅠ与XhoⅠ双酶切与pET-32a质粒连接,成功构建了大肠杆菌表达载体pET-32a-Ssp-Tα1,并转化大肠杆菌BL21 (DE3)菌株从而获得有效表达(目的蛋白占总蛋白50%以上)。我们通过一系列实验确认目的蛋白主要以可溶形式存在于菌体内,优化了表达条件(37℃, IPTG终浓度0.5 mM),并通过细胞破碎、收集离心上清、分子筛层析与Ni+亲和层析等过程纯化了重组蛋白。然后改变环境pH以诱导融合蛋白发生内切割,收集得到了N端无乙酰的Tal单体,酸性环境下(pH 3-4)使用醋酸酐对其进行体外乙酰化从而得到了完整结构的Tα1多肽,并对其进行HPLC纯化与质谱分析,验证了其分子量(3108 Da)。另外本实验通过western-blot,研究了Tal作用下HepG-2细胞内部分细胞周期调控因子的表达水平变化,发现Tα1能够诱导肿瘤细胞内Akt信号通路的阻滞和p53通路的激活,在降低cyclins与CDKs的表达水平的同时促进CDKI的表达,进而阻滞细胞周期于G1期。本实验在大肠杆菌表达系统中有效表达Tα1融合蛋白后,又通过内含肽诱导的蛋白自剪切技术得到Tα1单体,简化了Tα1的生产工艺并有望降低其成本,为大量生产Tα1满足临床需求开辟了新途径。同时发现Tal能通过调控细胞周期调控因子的表达水平来阻滞肿瘤细胞的生长周期,为进一步研究Tα1的生物功能提供了新的思路,也为Tal临床应用于肿瘤治疗提供了新的理论依据与数据支持。更多还原

【Abstract】 Human thymosin al (Tα1) is a biological polypeptide composed of 28 amino acid residues with immune-enhancing biological activity. It has widely functions such as enhancing the immune system, anti-tumor, endocrine regulation, and so on. At present, it is mainly used to cure chronic hepatitis B, hepatitis C clinically and as a powerful anti-virus drug for the treatment of bacterial or fungal infections, immunodeficiency, cancers, lupus erythematosus, etc. Thymosin alpha 1(Tα1) is so important, that it is meaningful to study either the expression or the function of this peptide. In this study, we transferred the Tal gene into E. coli to express it and make some chemically modification, and explored its role of tumor cell cycle arrest.First of all, we designed and synthesized the Tα1 gene according to the E. coli codon usage preference and constructed the with the intein self-shearing sequence concatemer gene (Ssp). We connected it to pET-32a and successfully constructed pET-32a-Ssp-Tal, transformed into E. coli BL21 (DE3) to make it expressed effectively (recombinant protein was above 50% of total protein). We confirmed that the target protein expressed in bacteria with soluble form and optimized the expression conditions (37℃, IPTG concentration is 0.5 mM), and through a series of processes (such as bacterial broken, centrifugal, Gel chromatography and Ni+ affinity chromatography), we purified the recombinant protein. We inducted it to self-shear through changing the pH and release the Tal monomer, then acetylated the Tal peptide with acetic oxide in vitro. HPLC separated the productions and Mass spectrometry revealed the weight of monomer was same with the nature Tal (3108 kD). In addition, we studied the expression levels of some cell cycle regulatory factors after HepG-2 cell be treated by Tal by western-blot, the results showed that Tα1 can reduce the Akt path and activate the p53 path, and can block the cell cycle by regulating the expression of cell cycle regulatory factors (cyclins and CDKs down while CDKI up) in the intracellular level. In this study, we built the fusion protein and expressed it in E. coli, got the Tal monomer by the intein self-shearing, so that the simple operations and the low costs which can be expected will make it possible to product Tal with large scale to meet clinical needs. We also found that Tα1 can regulate the expression level of some cell cycle regulators to inhibit tumor growth. And it provided an insight into the Tal-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Tα1 as an anti-tumor agent.更多还原