【作者】 关良；

【导师】 刘少君；

【作者基本信息】 中国人民解放军军事医学科学院 ， 神经生物学， 2002， 博士

【摘要】 肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL,也称为Apo-2L)是Wiley 1995年首次克隆成功的一种新的抗肿瘤细胞因子,属于TNF家族的一个成员,在人体的多种组织中如脾、肺、前列腺、胸腺和外周血淋巴细胞等正常组织均可发现其表达。人TRAIL蛋白编码281个氨基酸,它的基因位于染色体3q26。TRAIL与目前发现的TNF家族的其它主要成员,如TNF-α,TNF-β(也称为淋巴毒素或LT-α),淋巴毒素-β(LT-β),41BBL,OX40L,CD27L,CD30L,CD40L和FasL(CD95L,也称为Apo-1L)等配体均为Ⅱ型膜蛋白,可水解形成可溶性同源三聚体,参与调节机体内的许多重要生物功能。TRAIL除了在免疫调节和炎性反应方面起重要的作用外,还与TNF家族中的TNF-α、TNF-β、FasL配体可导致易感细胞凋亡。TRAIL与这些配体的区别在于,除可特异性导致肿瘤细胞发生凋亡外,不损伤正常细胞。这些细胞因子的跨膜受体含有死亡结构阈,与配体结合导致引起凋亡的一系列蛋白酶反应。本研究首先对合成的TRAIL寡核苷酸全长进行了诱导表达和纯化,获得了有活性的TRAIL蛋白,并验证其抗肿瘤效应。同时采用分子模建方法,对TRATL结构进行了缺失突变,得到了两个突变体,并对它们进行了热诱导表达,以便深入的研究结构与功能的关系,为TRAIL开发成新的抗肿瘤药物打下基础。 1.人TRAIL寡核苷酸全长合成及在大肠杆菌中表达和纯化 人TRAIL寡核甘酸全长843个碱基,我们对其分段合成,然后进行全长拼接,并改变其中的30个密码子,以纠正氨基酸偏性,更利于在原核载体中进行表达。核苷酸自动测序结果,与预想的序列一致。合成的产物亚克隆至pGEX-2T质粒构成TRAIL-pGEX-2T,然后转染受体菌DH-5α大肠杆菌。我们在低温30℃,用IPTG 0.4mM/L诱导TRAIL融合蛋白表达,得到了TRAIL融合蛋白。然后采用亲和层析方法纯化TRAIL蛋白。 2.TRAIL的抗肿瘤生物活性 用A172人胶质细胞瘤,B104神经母细胞瘤,SKNSH人神经母细胞瘤,更多还原

【Abstract】 TRAIL/Apo2 (TNF-related apoptosis inducing ligand), a novel cytokine, is a member of the tumor necrosis factor(TNF) family and was first cloned and identified by Willy in 1995, which can be detected in a varity of human tissue, most predominantly in spleen, lung, prostate,thymus and peripheral blood lymphocytes. TRAIL consists of 281 amino acids, and its gene is located on chromosome 3 at position 3q25. TRAIL as well as other members of TNF family, including TNF α , lymphotoxin β (LT β ),and ligands for CD40, CD30, CD27,OX40L is type II membrane protein, which can be hydrolyzed to soluble homotrimers. TRAIL play an important role in regulating many biological functions, such as prominent mediators of immune regulation and inflammatory responses, especially in inducing apoptosis in a wide variety of transformed cell lines and tumor cells. The key point between TRAIL and other apoptosis inducing ligands of the TNF family is that TRAIL effectively kills many tumor cells by apoptosis while leaving normal cells unharmed. These cytotoxic lignads induce the apoptosis (programmed cell death) by binding the receptors, which contain death domains.The full-length of TRAIL oligonucleotide was synthesized, and prokaryotic expression vector was constructed. After gene transfecting, the stable expression E coli DH-5α were obtained. Fusion TRAIL protein was expressed in E coli DH-5 α and purified by affinity chromatography. Subsequently, the purified protein wastested to assess its ability to induce apoptosis in various tumor cell lines such as BGC832 lung cancer and A172 glioma cells. We also got two TRAIL mutants through homology modeling. These two TRAIL mutants were expressed in E coli DH-5 α ,after their oligonucleotide, fragments were inserted into pBV220, a thermo-sensitive expression vector.The synthesis of full-length of TRAIL oligonucleotides, expression of TRAIL in E coli DH-5α and purification of TRAIL: The full-length of human TRAIL oligonucleotide codes for 841 bases, which was synthesized and thirty of which were replaced to make TRAIL expressed in E coli DH-5α efficiently. The DNA sequence analysis proved the full-length of human TRAIL oligonucleotide synthesized was the same as which we wanted. The mutant TRAIL oligonucleotide was inserted into pGEX-2T vector, which contains a GST protein code to be purified easily by affinity chromatography, then transfecting E coli DH-5 a . The fusion TRAIL protein was expressed in E coli DH-5 a induced by 0.4mM/L IPTG for 4 hours at 28℃, and purified by affinity chromatography through GSTrap column.Antitumor activity of TRAIL: The cytotoxic activities of TRAIL were measured on A172 huanm glioma cells, B104 murine neuroblastoma, SKNSH human neuroblastoma, BGC823 stomach cancer cells, A549 lung cancer cells, H69 lung cancer cells, KB nasopharyngeal cancer cells, etc. TRAIL to a final concentration of 1μg/ml is capable to induce apoptosis of these tumor cell lines efficiently.TRAIL mutant design based on homology modeling: TRAIL belongs to a member of TNF family, which gene codes for a polypeptide of 281 amino acids. The soluble active extracellular segment of TRAIL ranged from amino acids 95-281. The two TRAIL mutants were obtainedby homology modeling, one is residues 130-145 and 210-281, and the other is residues 145-281. The primers were designed according to oligonuclotide sequence of TRAIL mutants, 5’ primer with EcoR I restriction enzyme site and 3’ primer with BamH I restriction site. The mutants oligonucleotide segments were obtained by PCR, and then inserted into pUC18-T vector. The result of sequence analysis of mutants proved properly.Expression and purification of TRAIL mutants: The mutants oligonucleotide segments were inserted into pBV220.The recombinant plasmid pBV-TRAIL mutant identified by digestion with EcoR I AND BamH I was transformed to the competent cells of E coli DH-5α , and then the transformed cells were induced to be expressed by shifting culture from 37℃ to 42℃. The TRAIL mutants were expressed in the form of inclusion bodies and soluble protein. The inclusion bodies were washed with 8M urea, and then purified preparatively.更多还原