人α1微球蛋白质的克隆、表达、纯化及生物学活性鉴定

Clone,expression and purification of human α1-microglobulin as well as its bioactivity identification

摘要:

目的：构建人α1微球蛋白质（α1-microglobulin, α1M）基因的原核表达质粒，在大肠杆菌中表达α1M蛋白质，纯化并初步鉴定其生物学活性。方法：PCR扩增α1M基因序列，然后转化入大肠杆菌B834中进行表达，利用镍离子亲和层析、离子交换层析及分子筛纯化，ABTS法鉴定其生物学活性。结果：正确构建了α1M的表达质粒，α1M在大肠杆菌B834中为可溶性上清表达，纯化获得了纯度高达95%的α1M蛋白质，分子筛结果显示其在溶液中呈现单体和二聚体2种聚集状态，ABTS法结果显示了重组获得的α1M具有抗氧化活性。结论：α1M能在大肠杆菌B834菌中大量上清表达，且易于纯化，初步鉴定具有抗氧化活性，为下一步进行α1M蛋白质的晶体结构解析和相关功能研究奠定了基础。

Abstract:

Objective: To construct the expression vector of human α1-microglobulin (α1M) gene, express α1M protein in Escherichia coli (E.coli), purify recombinant α1M protein and to verify its bioactivity. Methods: The α1M gene sequence was amplified by PCR and α1M protein was expressed in E.coli B834. The recombinant protein was purified by nickel column chromatography, ion-exchange column chromatography and size exclusion chromatography and its bioactivity was verified by ABTS method. Results: The expression vector of the α1M was successfully constructed by PCR and α1M protein was expressed by E.coli B834 in a soluble supernatant way. The α1M protein with 95% purity was acquired successfully by purification. The result of the size exclusion chromatography indicated that α1M protein exhibited as both monomer and dimer in the solution. Additionally, the recombinant protein exerted anti-oxidant activity by ABTS method. Conclusion: α1M could be highly expressed in supernatant solution in E.coli B834 strain and it can be easily purified. The recombinant protein exhibits anti-oxidant activity, laying a solid basis for further structural and functional study of the human protein.