【作者】 王伟；

【导师】 陈正望；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 人异体移植炎症因子1( human allograft inflammatory factor 1,hAIF-1,Daintain)是上世纪90年代中后期欧美几个研究小组从不同的研究目标中鉴定出的一个由巨噬细胞和活化的T淋巴细胞产生的细胞因子。全长147个氨基酸,分子量17kDa。分子内含有一个Ca2+结合的EF-手形结构域,推测在细胞钙代谢中起一定作用,另有一个由44个氨基酸残基片断组成的激素前体序列模式KR-KK-GKR,对胰岛素的分泌具有双向调节作用。不同的实验室研究发现,Daintain/AIF-1在肿瘤的发生和发展,心脏、肝脏的移植排斥,中枢神经系统损伤以及自身性免疫疾病中发挥重要作用。由于其含义深刻的结构和重要的生物学功能,已引起了生命科学和医学界的广泛关注。由于Daintain/AIF-1在自然界中丰度很低,从天然产物中提取大量Daintain/AIF-1非常困难。所以本研究以Daintain/AIF-1氨基酸序列为依据,人工合成基因寡核苷酸片段,通过质粒抽提,酶切,将含Daintain/AIF-1的片断克隆到原核表达载体pET32-a(+)中,得到重组质粒pET32-a(+)-AIF-1,然后转化大肠杆菌BL21(DE3)。通过对诱导表达条件的优化,30℃,IPTG诱导2h后可溶性表达。经Sephrose-镍亲和层析柱纯化,并由SDS-PAGE鉴定产物的表观分子量约1.8×104Da。Western boltting进一步证明纯化产物含Daintain/AIF-1。说明Daintain/AIF-1-6His在大肠杆菌BL21(DE3)中表达成功。通过该方法来表达和纯化Daintain/AIF-1的突出优点是流程短,成本较低,技术成熟,可靠性高。该工作为Daintain/AIF-1的结构和功能研究打下了基础,对以后用基因工程方法生产该蛋白,并用于临床具有一定意义。更多还原

【Abstract】 Allograft inflammatory factor 1 (AIF-1)/ Daintain is a 147-aa residue polypeptide with a mass of 17kDa,characterd as a macrophage and T Lymphocyte factor by several European and American searth teams in the midanaphase of 90’s last century for different aims . Daintain/AIF-1 has an Ca2+-binding EF-hand suggested some function in Ca2+ metabolism,and an internal 44-residue segment with the sequence pattern–KR–KK–GKR–, a motif typical of peptide hormone precursors, regulating insulin secretion. Daintain/AIF-1 has been verified an important protein in tumor progress, heart/liver transplant rejection, central lesion, and autoimmune disease etc. For interesting structure and function, Daintain/AIF-1 is widespread investigated in biology and medical science.Daintain/AIF-1 is too little to purify from wild material. So its oligo-nucleotide fragment was artificially synthetic based on the amino-acid sequence of Daintain/AIF-1 . After extracting plasmid, enzyme hydrolysis,the fragment was cloned into prokaryotic expression vector pET32-a(+). The recombinant expression plasmid pET32-a(+) -Daintain/AIF-1-6His was transformed into E.coli BL21(DE3). Then the conditions of inducement were optimized at 30℃, 2h. The fusion protein Daintain/AIF-1-6His was solubly expressed. The fusion protein was purified by Sephrose-NI affinity column. The molecular mass of the fraction determined by SDS-PAGE was about 18kDa, Existence of Daintain/AIF-1 was further identified by Western blotting .So the Daintain/AIF-1-6His fusion protein was successfully expressed in E. coli.The work provides preparation for further study of the function of Daintain/AIF-1 and producing the protein with gene engineering for clinical application.更多还原