ZZ亲和肽-碱性磷酸酶融合蛋白的制备与初步应用  

Preparation of fusion protein between ZZ affinity peptide and alkaline phosphatase and its primary application

作　　者：鲍如梦 赵爽佳 薛敏[1] 杨洪鸣[1] 唐金宝[1] 

机构地区：[1]潍坊医学院药学院,潍坊261053

出　　处：《中国免疫学杂志》2015年第9期1214-1216,1220,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.81201346);山东省自然科学基金(No.ZR2013HL066)基金资助

摘　　要：目的:构建、表达ZZ亲和肽与碱性磷酸酶(AP)融合蛋白,并研究其生物学活性。方法:将ZZ亲和肽基因与AP基因重组,构建原核表达载体p EZZ-AP并利用大肠杆菌表达,金属离子亲和层析纯化目的蛋白,Western blot鉴定ZZ-AP生物学活性并初步应用于免疫细胞化学。结果:所构建重组质粒p EZZ-AP经E.coli DH5α表达,SDS-PAGE结果显示目的蛋白相对分子量为62 k D,与理论值相近。Western blot结果表明ZZ-AP融合蛋白既具有兔Ig G抗体结合活性,又具有碱性磷酸酶活性,在细胞免疫组化应用中与AP标记的羊抗兔二抗显色模式与效果相似。结论:ZZ-AP融合蛋白构建成功,该融合蛋白具有兔Ig G抗体结合活性和碱性磷酸酶活性,细胞免疫组化应用结果表明该融合蛋白在免疫分析中具有潜在的应用价值。Objective：To construct and express the fusion protein between ZZ affinity peptide and alkaline phosphatase and examine its biological activities.Methods：The alkaline phosphatase gene was cloned into pEZZ 18 vector containing ZZ peptide gene resulted in the pEZZ-AP recombinant vector.Then the vector was transformed and expressed in E.coli DH5α.And HisTrap affinity chromatography was employed to separate and purify the target protein.After analyzed by Western blot , ZZ-AP fusion protein was applied to immunocytochemistry as an alterative second antibody.Results：The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values （62 kD）.The fusion protein has rabbit IgG-binding ability and en-zymatic activity of alkaline phosphatase ,those were validated in Western blot;and it produced a good signal that was comparable in its staining pattern to that generated with goat anti-rabbit IgG-AP in immunocytochemistry.Conclusion： The ZZ-AP fusion protein was constructed successfully ,it has rabbit IgG-binding ability and enzymatic activity of alkaline phosphatase.We anticipate that the ZZ-AP fusion protein has a potential application in immunoassay field.

关 键 词：ZZ亲和肽 碱性磷酸酶 融合蛋白 应用 

分 类 号：Q591.2[生物学—生物化学]