人可溶性增殖诱导配体在大肠杆菌中的表达、纯化及生物活性鉴定  

Cloning, Expression and Characterization of Human Soluble APRIL in Escherichia Coli

作　　者：岳园园[1] 水燕[1] 管政兵[1] 张双全[1] 

机构地区：[1]南京师范大学生命科学学院,江苏省分子医学生物技术重点实验室,江苏南京210097

出　　处：《南京师大学报（自然科学版）》2006年第4期86-90,共5页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30271093).

摘　　要：人增殖诱导配体在促肿瘤细胞增殖以及自身免疫性疾病中具有重要的作用.从GenBank中查出人APRIL的序列号(AF046888),根据其胞外可溶部分序列设计引物,采用RT-PCR技术从健康人新鲜血液单核细胞(PBMCs)总RNA中克隆出人sAPRILcDNA,将该cDNA克隆入原核表达载体pET30a,经IPTG诱导后在大肠杆菌BL21(DE3)中获得高效表达,经SDS-PAGE后在17000左右有一明显的表达条带,该蛋白主要以包涵体形式存在,表达量达45%左右,将包涵体蛋白变复性后进行细胞生物活性实验,为进一步研究其临床应用奠定了基础.A proliferation-inducing ligand plays an important role in promoting the proliferation of tumor cells and its own immunized diseases. We screen the accession of hsAPRIL in GenBank （ AF： 046888 ） and design the primers according to part of its extracellular sequence. The total RNA , extracted from human blood , is amplified into the sequence of eDNA encoding sAPRIL by RT-PCR and cloned into the vector pET30a for expression in Escherichia coll. After purification and refolding, the protein is used for biological activity assay. After induction by IPTG, the protein is examined by SDS-PAGE and its active form is obtained. After sequencing, the APRIL gene is confirmed to clone into the recombinant expression vector. The rhsAPRIL is about up to 40% in the total protein expressed in the Escherichia coll. After purification and refolding, the protein can stimulate tumor cell proliferation.

关 键 词：增殖诱导配体 基因克隆 肿瘤细胞 增殖 

分 类 号：R392.11[医药卫生—免疫学]