IL 1 5及IL 1 5R阳性细胞在成人T淋巴细胞性白血病 (ATL)、多发性骨髓瘤及炎症性自身免疫性疾病病理过程中起着重要作用 .应用基因重组技术 ,构建、表达靶向于IL 1 5受体的两种融合蛋白 ,为研制特异的可消除IL 1 5R高表达细胞的导向药物奠定基础 .将人IL 1 5成熟肽基因及IL 1 5R拮抗剂 (IL 1 5M)基因片段分别与人工改造的人肿瘤坏死因子突变体 (TNFαM)基因按正确的阅读框架融合 ,定向克隆在pET1 6b表达载体T7启动子的下游 ,得到质粒pET IL 1 5 TNFαM和pET IL 1 5M TNFαM .从大肠杆菌相应重组菌株中通过Ni2 + NTA亲和层析分别纯化出两种融合蛋白IL 1 5 TNFαM和IL 1 5M TNFαM .IL 1 5 TNFαM和IL 1 5M TNFαM对IL 1 5R阳性红白血病细胞K5 6 2的杀伤作用分别是TNFα的 4和 1 5倍 ,两种蛋白对IL 1 5R阴性细胞系Jurkat的杀伤作用则没有明显差异且均弱于TNFα .这些结果说明 ,两种融合蛋白特别是IL 1 5受体拮抗型蛋白IL 1 5M TNFαM对与IL 1 5 IL 1 5R异常表达相关的疾病可能具有潜在的治疗价值
Abstract
IL 15 and IL 15 receptors(IL 15R) play a crucial role in the development and progression of adult T cell leukemia(ATL), multiple myeloma and inflammatory autoimmune diseases. Recombinant DNA techniques were used to construct two targeted molecules that were designed to eliminate IL 15R overexpressing cells. A gene for human tumor necrosis factor mutant(TNFαM) was genetically fused to the DNA encoding wild type IL 15 or an IL 15 antagonist(IL 15M) and further cloned into pET16b under the control of T7 promoter, giving rise to pET IL 15 TNFαM and pET IL 15M TNFαM, respectively. Using Ni 2+ NTA affinity chromatography, IL 15/TNFαM and IL 15M/TNFαM were purified from E.coli BL21(DE3)plysS transformed with either pET IL 15 TNFαM or pET IL 15M TNFαM. The cytotoxicity of IL 15/TNFαM and IL 15M/TNFαM against IL 15R positive myelogenous leukemia cell line K562 was approximately 4 and 15 fold higher than that of recombinant TNFα, respectively. However, the cytotoxicity of both fusion constructs on IL 15R negative cell line Jurkat did not show marked difference and was relatively lower, compared to TNFα. The present data suggest that both protein fusions, especially IL 15M/TNFαM which is unable to trigger the activation of target cells, may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R.
关键词
肿瘤坏死因子 /
白细胞介素15 /
突变 /
融合 /
基因工程
{{custom_keyword}} /
Key words
tumor necrosis factor /
interleukin 15 /
mutation /
fusion /
genetic engineering
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
1 FehnigerTA ,CaligiuriMA .Interlukin15:biologyandrelevancetohumandisease.Blood,2001,97(1):14～32
2 PettitDK ,BonnertTP ,EisenmanJ ,SrinivasanS ,PaxtonR ,BeersC ,LynchD ,MillerB ,YostJ,GrabsteinKH ,GombotzWR .Structure functionstudiesofinterleukin15usingsite specificmutagenesis,polyethyleneglycolconjugation,andhomologymodeling.JBiolChem,1997,272(4):2312～2318
3 HansenMB ,NielsenSE ,BergK .Re examinationandfurtherdevelopmentofapreciseandrapiddyemethodformeasuringcellgrowth cellkill.JImmunolMethods,1989,119:203～210
4 AndersonDM ,KumakiS ,AhdiehM ,BertlesJ ,TometskoM ,LoomisA ,GiriJ,CopelandNG ,GilbertDJ ,JenkinsNA ,ValentineV ,ShapiroDN ,MorrisSW ,ParkLS ,CosmanD .Functionalcharacterizationofthehumaninterleukin15receptorαchainandcloselinkageofIL 15RAandIL 2RAgenes.JBiolChem,1995,270:29862～29869
5 WaldmannTA ,TagayaY .Themultifacetedregulationofinterleukin15expressionandtheroleofthiscytokineinNKcelldifferentiationandhostresponsetointracellularpathogens.AnnuRevImmunol,1999,17:19～49
6 ChaSS ,KimJS ,ChoHS ,ShinNK ,JeongW ,ShinHC ,KimYJ ,HahnJH ,OhBH .Highresolutioncrystalstructureofahumantumornecrosisfactor alphamutantwithlowsystemictoxicity.JBiolChem,1998,273:2153～2160
7 KatsanisE ,XuZ ,PanoskaltsisMA ,WeisdorfDJ,WidmerMB ,BlazarBR .IL 15administrationfollowingsyngeneicbonemarrowtransplantationprolongssurvivaloflymphomabearingmice.Transplantation,1996,62(6):872～875
8 刘堰,胡应和,欧阳克清,蔡绍.突变型人白细胞介素2基因在巴斯德毕赤酵母中的表达.中国生物化学与分子生物学报(LiuYan,HuYing he,OuyangKe qing,CaiShao xi.Expressionofanewtypehumaninterleukin2geneinPichiapastoris.ChinJBiochemMolBiol),2003,19(5):618～624
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
PDF(350 KB)
