小鼠白细胞介素-15与其受体α亚单位协同作用的初步研究

Preparation of Mouse IL-15 and Study on Its Synergetic Effect with Mouse IL-15R α

目的原核表达小鼠白细胞介素-15（mouse interleukin 15，mlL-15），并初步研究其与受体α亚单位（mlL-15R α）的协同效应。方法从小鼠肾脏组织克隆mlL-15成熟从部分的基因片段，并亚克隆至原核表达载体pQE-30，所得重组表达质粒pQE-30/mlL-15转化EcoliM15。得到的转化子经IPTG诱导后获得重组表达产物，并进行SDS-PAGE和Western Blot检测。Ni-NTA亲和层析柱上纯化、复性重组蛋白，以获得成熟的mlL-15。采用MTT法检测重组mIL-15促CTLL-2细胞增殖的生物学活性，并进一步研究，mlL-15与mlL-15Rd结合后协同促进靶细胞增殖的功能。结果重组mlL-15丰要以包涵体形式存在，其相对分子质最约为14ku，并能为抗His和抗mlL-15抗体昕特异性识别。经Ni-NTA柱上蛋白纯化、包涵体复性后，可得到纯度约为95％的成熟蛋白。成熟mlL-15能够促进CTLL-2细胞增殖，并具有明显的屋效关系；并且，在mIL-15Rα的配合下，mlL-15的促CTLL-2细胞增殖的活性冠著增强，尤其足在低mIL-15浓度条件下该协同作用表现得愈发明显。结论获得了具有牛物活性重组mlL-15，并初步研究了其与mlL-15Ra组成复合物的体外协同效应。该研究成果为进一步研究lL-15及IL-15Rd协同性的作用机理和开展小鼠成体免疫治疗研究奠定了基础。

Objective To express the mature protein of mouse interleukin 15 （mlL-15） in prokaryotic cells and study the synergetic effect of it with mlL-15R α in vitro. Methods The gene which encoding the mature peptide of mouse IL-15 was cloned using total RNA extracted from mouse kidney and then sub-cloned into pQE-30 expressing vector to form the reconstructed plasmid,pQE-30/mlL-1 5.The plasmid was then transfected into E.coli M 1 5 strain.The recombinant strain was induced by IPTG and the expression of mlL-15 was determined by SDS- PAGE and further identified by western blot.Affinity purification and refolding ofmIL-15 were performed on Ni- NTA column.For biological function analysis,raiL-15 was double diluted and then added to the culture medium of CTLL-2 cells.The cells proliferation was determined using MTT method.Further more,in order to investigate its synergetic effect with its unique receptor α subunit,titrated mIL-I 5 was mixed and combined with mIL-I 5R α at first,and then added to the culture medium. Results The recombinant mlL-15 had a molecular mass of about 14 ku and mainly expressed as inclusion body.It could be specifically identified by anti-His and anti-lL-15 monoclonal antibody,respectively.After Ni-NTA affinity purification and refolding,mIL-15 with the purity more than 95% was obtained.There was a clear dose-response relationship between the CTLL-2 cells proliferation and the raiL-15 concentration.Interestingly,the stimulation effect of raiL-15 was significantly enhanced at the existence of miL- 15R a ,especially at the lower mlL-15 concentration. Conclusion We successfully prepare biofunctional recombinant protein mlL-15 and preliminarily investigate its synergetic effects with raiL-15R α in vitro.This makes it possible to further investigate the synergetic effects between mlL-15 and mlL-15R a systematically in vivo.