【作者】 陈玉辉；

【导师】 唐克轩；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 胸腺素α1(Thymosinα1,Tα1)是人体中枢免疫器官胸腺所分泌的一种免疫活性极强的28肽激素,具有促进T细胞分化和成熟,提高免疫的作用,还可以治疗自身免疫疾病,在临床上具有很好的效果。临床用Tα1主要靠从动物的胸腺组织中提取或进口国外的化学合成品,存在着组织来源有限、价格昂贵和安全性隐患等问题,使临床应用受限。因此本研究拟利用番茄作为表达系统表达Tα1,探索Tα1生产新途径。本研究根据植物偏爱密码子原则设计合成了Tα1,同时考虑到Tα1基因较小,在植物系统中表达易被降解的问题,利用基因工程和同尾酶技术将其串联成四联体,即4×Tα1。利用PG启动子构建了在番茄果实中特异表达融合蛋白4×Tα1的植物双元表达载体PG-pRD12-4×Tα1,利用冻融法转入农杆菌EHA105,通过农杆菌介导法转化番茄,经卡那霉素筛选,最终获得了再生植株54株。经PCR、Southern blot分析确认了其中4株独立的转基因植株。RT-PCR分析结果表明,在番茄的成熟果实和未成熟果实中均有目的基因4×Tα1的表达,而在叶片中没有表达。对转基因番茄果实蛋白进行了Western Blot分析,证明在转基因植株中成功表达了4×Tα1。番茄果实的ELISA定量分析显示,成熟果实中4×Tα1表达量介于2.725μg/g.FW至6.094μg/g.FW之间,而未成熟绿果中4×Tα1表达量介于2.373μg/g.FW至2.434μg/g.FW之间。小鼠淋巴细胞增殖试验证明转基因番茄中表达的4×Tα1蛋白具有生物活性,这是首次成功利用番茄果实特异表达系统表达具有生物活性4×Tα1的报道。虽然口服给药可以降低成本,使用方便,但口服给药剂量较难控制,临床仍以静脉注射给药为主。为了纯化番茄中表达的4×Tα1,本研究也对目的蛋白4×Tα1的纯化进行了相应的探索,以期为后续规模化纯化4×Tα1提供依据。根据原核表达载体生产重组蛋白具有操作简便、周期短,并且原核表达载体本身含有his-tag结构,便于利用Ni-NTA介质对目的蛋白进行分离纯化的特点,本研究还构建了含4×Tα1基因的原核表达载体pQE30-4×Tα1,随后转化大肠杆菌菌株M15,在IPTG的诱导下成功表达了目的蛋白。结果表明,根据植物偏爱密码子设计合成的4×Tα1基因,可以在大肠杆菌中高效表达。通过纯化得到了纯度较高的4×Tα1蛋白,所表达蛋白表现出了良好的生物活性。这是首次利用原核系统表达按植物偏爱密码子设计合成的Tα1基因的报道。该研究不仅为利用番茄系统表达4×Tα1基因提供了阳性蛋白的参照,同时也为今后利用植物系统表达目的蛋白的纯化提供了参考和借鉴。更多还原

【Abstract】 Thymosin alpha-1(Tα1) is an acetylated 28-amino-acid peptide hormone from the thymus glands of mammals. Thymosin plays important roles in modulating immune response and has significant clinical applications used as therapeutic agents. Tα1 has been evaluated for its immuno-modulatory activities and therapeutic potential in several diseases including chronic hepatitis B and C, acquired immunodeficiency syndrome, and depressed response to vaccination and cancer. Nowadays, the main methods to get thymosin are via extraction from animal tissues or via chemical synthesis. However, the high price confine the application of the thymosin. In recent years there has been a growing interest of producing therapeutic proteins and vaccines in transgenic plants. Plants offer the prospect of inexpensive biopharmaceutical production without sacrificing product quality or safety. It will be interesting to see if plants can be used to produce Tα1, by which the cost and complication to produce Tα1 may be lowed down. In this study, Tα1 gene was designed and synthesized according to plant codon usage preference. The tandem Tα1 gene of 4 repeats was concatenated and identified by sequencing. A plant expression vector PG-pRD12-4×Tα1 harboring the 4×Tα1 gene was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 54 putative transgenic tomato plants were regenerated. PCR, Southern blot analyses confirmed the transgenic status of some plants. RT-PCR analysis demonstrated that the 4×Tα1 gene was expressed specifically in tomato fruits. Western blot analysis confirmed the expression of the 4×Tα1 concatemer protein in tomato fruits. ELISA analysis showed that the concentration of the 4×Tα1 concatemer protein ranged from 2.725~6.094μg/g fresh weight in mature fruit. However in the immature fruits, the concentration of the 4×Tα1 concatemer protein ranged from 2.373~2.434μg/g fresh weight. This study confirmed that the 4×Tα1 concatemer protein was successfully expressed in tomato. Biological activity of expressed 4×Tα1 concatemer protein was carried out by lymphocyte proliferation assay, and the result demonstrated that the protein had effects of immuno-modulation. This is the first report on successful expression of biologically active thymosin alpha 1 (Tα1) in plants and this research provides a basis for further development of plants as bioreactor to produce Tα1, one of the important therapeutic proteins. Although the oral immunogenic protein antigens can low down the production cost, it is difficult to control the oral therapy dose. The mainline is also widely applied in clinic. Therefore the purification of target protein from the transgenic plants was very important. In this study, some researches about protein purification were carried out. Considered the advantage of Escherichia coli (E. coli) expression system in expression time, the E. coli expression vector pQE30-4×Tα1 containing 4×Tα1 gene was constructed which was subsequently transformed into E. coli M15. Vector pQE30 was used because the target protein could be expressed as a fusion protein with a 6×His-tag for the convenience of subsequent purification. The result showed that the 4×Tα1 gene was successfully expressed in E. coli. The expressed protein was purified and found to have the normal bioactivity. This is the first report on the expression of 4×Tα1 concatemer which is synthesized according to plant codon usage preference in E. coli expression system. This study not only provides the positive protein of 4×Tα1, but also provides the reference in purification of expressed protein in plants in the future.更多还原