【作者】 王浩；

【导师】 邵健忠；

【作者基本信息】 浙江大学 ， 细胞生物学， 2010， 硕士

【摘要】 白细胞介素-1(Interleukin-1,IL-1)是在局部和全身炎症反应中起关键作用的细胞因子,包括IL-1α和IL-1β(又分别被命名为IL-1F1和IL-1F2)。目前,IL-1家族在哺乳动物中共发现了11个因子,包括IL-1F1-IL-1F11,但是在鱼类或其他低等脊椎动物中,除IL-1β、IL-18和IL-1RA之外的家族成员尚未被发现。IL-1的生物学效应通过结合其受体,即IL-1受体(IL-1 Receptor)后的信号转导来实现。IL-1受体家族包括Ⅰ型受体(IL-1R1)、Ⅱ型受体(IL-1R2)和IL-1受体辅助蛋白(IL-IRAcP)。本文利用生物信息学技术,在河豚鱼(Tetraodon nigroviridis)中,成功地预测并克隆了IL-1β基因和IL-1R1样基因(Tetraodon IL-1R1-Like),并且利用DNA重组技术构建了TnIL-1β原核表达载体,转化表达菌株BL21,经IPTG诱导,成功表达并纯化到TnIL-1β蛋白,制备了TnIL-1β兔抗血清。TnIL-1β含有4个外显子,3个内含子,开放阅读框(ORF)长度为774bp,编码257个氨基酸。启动子区域含有TATA Box、NF-KB、CEBP、AP1、CCAAT等转录因子结合位点等,3’UTR含有5个ATTTA instability motif。将TnIL-1β的ORF亚克隆到原核表达载体pET41a上,成功构建了重组表达载体TnIL-1β-pET41a。用IPTG诱导使表达菌株BL21大量表达TnIL-1β蛋白,Ni-NTA纯化后能够得到分子量为60.1Kd的纯化条带。TnIL-1R1-Like基因共有11个外显子,ORF长度为1902bp,编码633个氨基酸,含有3个Ig Domain,1个跨膜区,1个TIR Domain。综上,本研究为后续开展鱼类IL-1和IL-1R的功能和相互作用关系研究奠定了基础,对研究鱼类免疫系统功能和基因进化具有重要的意义。更多还原

【Abstract】 Interleukin-1 plays an important role in the local and systemic inflammatory response;including IL-1αand IL-1β(named as IL-1F1 and IL-1F2,respectively).At present,the Interleukin-1 family arised in vertebrates consists of 11 known genes encoding ligands(IL-1F1 to IL-1F11).But to date,no orthologue to IL-1βis found in fish or other low vertebrates.IL-1 functions by binding their receptors,IL-1 Receptor,to mediate signal transduction to realize their biological effects.IL-1 Receptor family contains typeⅠreceptors(IL-1R1),typeⅡreceptors(IL-1R2),and the IL-1 receptor accessory protein(IL-1RAP).In this study,we developed bioinformatics methods based on genomic databases, successfully cloned IL-1βgene and IL-1R-like gene in Tetraodon,and obtained recombinant TNIL-1βprotein and rabbit anti-IL-1βserum.TNIL-1βcDNA contains 4 exons,3 introns and a 774bp open reading frame(ORF).The peptide deduced from ORF is 257aa in size.TNIL-1βpromoter comprises several transcription binding sites, such as TATA-box,NF-KB,CEBP,AP1,CCAAT,and so on,3’UTR contains 5 ATTTA instability motif.A recombined plasmid TNIL-1β-pET41a was obtained by sub-cloning the ORF of TNIL-1βinto prokaryotic expression plasmid pET41a and sequenced.Target 60.1Kd protein was expressed after IPTG induce and purified by Ni-NTA.TNIL-1R-like gene contains 11 exons,and a 1902bp ORF,coding 633aa, which has 3 Ig Domain,1 transmembrane domain(TMD),and 1 TIR Domain.All the results lay foundation in function studies of fish IL-1βand IL-1R,and is important for fish immune system research and evolution of immunology genes.更多还原