鸡Tie-2受体胞外片段的原核表达及目的蛋白免疫原性的研究  

Prokaryotic Expression, Purification and Refolding of Extracellular Ligand Binding Domains of Chick Tie-2 and Its Immunogenicity

作　　者：罗彦[1] 田聆[1] 吴扬[1] 李秋[1] 刘继彦[1] 胡兵[1] 宿静梅[1] 何秋明[1] 刘成文[1] 魏于全[1] 文艳君[1] 

机构地区：[1]四川大学华西医院人类疾病生物治疗教育部重点实验室肿瘤中心

出　　处：《四川大学学报（医学版）》2004年第2期154-157,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金重点项目 (批准号 3 0 13 0 2 6)资助

摘　　要：目的　研究鸡 Tie- 2受体胞外片段 (配体结合域 )的原核表达以及目的蛋白对小鼠的抗原性。方法　用 PCR方法从既往构建的包含鸡 Tie- 2受体胞外段基因的表达质粒扩增目的片段 ,进而构建 PQE原核表达载体并诱导其目的蛋白的表达 ,将目的蛋白进一步纯化、复性并免疫小鼠 ,获得抗血清 ,用 EL ISA和 Western blotting检测抗血清中抗体的存在。结果　经酶切分析及测序鉴定表明获得了鸡 Tie- 2受体胞外目的片段的 PQE原核表达载体 ,能高效表达目的蛋白 ,将其免疫小鼠可产生抗重组蛋白的抗体。结论　用原核表达的方式可成功的获得鸡Tie- 2受体胞外目的片段的蛋白 。Objective To study the prokaryotic expression of extracellular ligand binding domains of chick Tie-2, the purification and refolding conditions of the recombinant protein, and to observe its immunogenicity in mouse. Methods A DNA fragment encoding extracellular ligand binding domains of chick Tie-2 was obtained by PCR from a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector PQE30, and was expressed in E.coli XL-1 blue by adding isopropyl-β-D-thiogalactoside(IPTG).The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected into mouse subcutaneously, and the antiserum of the mouse was analyzed by ELISA and Western blot analysis. Results The recombinant protein was highly expressed in E.coli XL-1 blue, and in mouse it produced the antibody which could specifically recognize the recombinant protein. Conclusion The protein of extracellular ligand binding domains of chick Tie-2 can be highly expressed in prokaryotic expression system, and the expressed protein can induce immune response in mouse. These findings are very important for the further study of this protein in anti-angiogenesis and immunotherapy research.

关 键 词：鸡 TIE-2受体 胞外片段 原核表达 蛋白免疫原性 抗体 血清 

分 类 号：R392[医药卫生—免疫学]