【作者】 孙丽敏；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 遗传学， 2007， 硕士

【摘要】 目的：穿膜肽是指具有细胞膜穿透功能、长度多数小于20个的氨基酸序列，可以穿过细胞膜进入细胞质甚至细胞核，而细胞膜却完好无损。这些天然的或人工合成的多肽具有水溶性、低裂解性并通过非吞噬作用进入各种细胞膜。其中发现较早，研究最多的是来源于人免疫缺陷病毒（HIV）-Ⅰ的反式转录活化因子（Trans—activating transcriptional activator，TAT）。TAT或TAT来源的多种多肽不仅能运输小分子物质，还能运输大分子量的药物和纳米颗粒性物质至多种哺乳动物的活细胞中。Rhox基因家族是一簇新发现的同源异型框基因簇，其定位于X染色体上，特异性的在雌雄鼠的生殖组织中表达，可能在胚胎发育、生殖组织的发育、精子的生成和成熟中发挥重要作用。本实验室利用GAL4酵母双杂交系统对小鼠7天胚胎cDNA文库进行筛选，获到了几个与mPem蛋白相作用的分子，其中包括Cdc37和Mdfic。本课题拟构建，表达和纯化TAT-Rhox5融合蛋白，并将纯化所得蛋白质传导进入细胞，研究穿膜肽TAT是否可以协助Rhox5蛋白进入细胞和其在细胞内的定位，以及其在细胞内的功能发挥情况。从而为穿膜肽TAT的应用研究提供实验依据，同时，为Rhox5蛋白的功能研究奠定基础。方法：将TAT序列按照通读框插入重组质粒pET22b（+）-Rhox5中，构建pET22b（+）-TAT-Rhox5表达质粒。转化E.coli表达菌株Rosseta^TM2（DE3），IPTG诱导表达TAT-Rhox5-6His融合蛋白，Western blotting检测目的蛋白，表逹并纯化TAT-Rhox5融合蛋白，转导TAT-Rhox5融合蛋白进入细胞并检测其转导进入细胞的最佳浓度，再以免疫荧光法检测其在细胞内的定位。结果：成功构建pET22b（+）-TAT-Rhox5原核表达质粒；实现了TAT-Rhox5-6His蛋白在大肠杆菌中的高效表达；并能纯化出高纯度的TAT-Rhox5融合蛋白，证实穿膜肽TAT可以协助Rhox5蛋白进入细胞，并最终运送到核内，而TAT-Rhox5融合蛋合进入细胞的最佳浓度为3μM，当转导时间增加，融合蛋白进入细胞核的量亦相对增加。结论：成功纯化出高纯度的TAT-Rhox5融合蛋白，证实穿膜肽TAT可以协助Rhox5蛋白进入细胞，并最终运送到核内，为Rhox5蛋白的功能研究奠定基础。更多还原

【Abstract】 Objective: The penetrating peptides, usually no more that 20 amino acids, are able topenetrate the cellular membrane including the nuclear evelope without damaging it. This naturalor artificial polypeptides, which are water soluble and of low spallation, can also penetratevarious cellular membranes through the inphagocytosis. Amongst them, the Trans-activatingtranscriptional activator of the Human immunodeficiency virus, TAT, was studied intensively.TAT or its derivatives can transport both micromolecule substance and macromolecule drugs, aswell as nanometer substance to difference living mammalian cells. Rhox gene family is a newhomeobox gene cluster which allocates on the X chromosome and expresses only in female andmale mice reproduction system. Despite its potential role in regulating the embryonicdevelopment, particularly the development of germinal tissue and the production and maturity ofsperm, the cellular function of Rhox5 was left largely unknown. In previous study, wedemonstrated interactions between Rhox5 and mPem,. Cdc37 and Mdfic in a yeast two-hybridscreen of a 7-day mouse embryo library. In this study, a TAT-Rhox5 fusion protein wasexpressesed and purified in E.Coli for further functional study of Rhox5.Method: The sequence encoding TAT was cloned into pET22b（+）-Rhox5 in frame of Rhox5.The resulted pET22b（+）-TAT-Rhox5 was transformed into Rosseta^XM2（DE3）. The expression ofTAT-Rhox5-6His fusion protein was induced by IPTG and verified by Western blot assay. TheTAT-Rhox5 was purified and used to transduct COS-7 cells subsequently. The intracellularlocalization of TAT-Rhox5 was observed by immumofluorescence.Result: The pET22b（+）-TAT-Rhox5 prokaryotic expression vector was constructedsuccessfully and the TAT-Rhox5-6His fusion protein was highly expressed in E. coil. Thepurified TAT-Rhox5 fusion protein was uptaken by NIH3T3 cells. Except a small amount innucleus, TAT-Rhox5 showed a cytoplasmic distribution in the tested cells. The best contration oftransduce of TAT-Rhox5 fusion protein is 3μM. When the transduce time was increase, there aremore fusion protein distribution in nucleus.Conclusion: The TAT-Rhox5 fusion protein was purified successfully and it was uptaken byNIH3T3 cells. TAT-Rhox5 fusion protein was distribution in cytoplasmic and nucleus, the resultwas settling a foundation for further functional study of Rhox5.更多还原