摘要
目的在大肠杆菌中表达人IL-21编码基因,并进行蛋白纯化和抗原性分析。方法以经PHA刺激的人扁桃体细胞cDNA文库为模板,经PCR获得IL-21全基因片段。测序确认后,分别设计含BamHⅠ和SalⅠ酶切位点的引物,经PCR获得IL-21成熟肽的编码基因。将此IL-21基因插入表达载体pGEX-4T-2,重组克隆经酶切与测序确认后转化大肠杆菌DH5α,进行IPTG诱导表达。经琼脂糖珠结合的纯化方法所得产物用WesternBlotting作抗原性分析。结果SDS-PAGE显示经IPTG诱导后的大肠杆菌DH5α表达与理论相符的条带,并获得了与理论值相符的纯化条带,在进一步的Western Blotting分析中发现表达的蛋白能与IL-2的单抗产生结合反应。结论人IL-21编码基因克隆载体在大肠杆菌中成功表达,同时建立了该蛋白的琼脂糖珠纯化方法,通过免疫印迹实验揭示了原核表达的IL-21蛋白与IL-2之间结构的相似性。
Objective To express coding gene of human interleukiw21 （IL-21） in E. coli, purify its protein, and assay its antigenicity. Methods Human IL-21 gene fragments were obtained from human tonsil cDNA by PCR. The coding gene for the mature N terminus of IL-21 was inserted into prokaryotic expressing vector pGEX-4T 2 and transformed into E. coli DH5α. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead and its antigenicity analyzed by Western Blotting. Results Induced by IPTG, E. coli DH5α expressed the right molecular weight protein. Western Blotting test showed the protein bad reaction with IL-2 monoclonal antibody. Conclusion The vector pGEX-4T-2/IL-21 and engineering bacteria E. coli DH5α expressing IL-21 mature N terminus fusion protein is successfully constructed. By purification and Western Blotting test, the recombinant IL-21 revealed resemblant antigenicity to IL-2.
出处
《青岛大学医学院学报》
CAS
2008年第1期41-43,共3页
Acta Academiae Medicinae Qingdao Universitatis
基金
青岛市科技局科研基金项目(04-2-JZ-95)
滨州医学院科研启动资金资助课题(BY2005KYQD02)
