β-半乳糖苷酶供体片段定点突变、表达、纯化与活性分析

Site-specific mutagenesis,expression,purification and complementation analysis of β-galactosidase donor

目的构建含ε-NH2标记位点的克隆酶供体免疫分析（CEDIA）系统供体片段。方法从大肠杆菌基因组克隆β-半乳糖苷酶（β-gal）的α片段基因,通过定点突变在原精氨酸位点处引入赖氨酸突变,与硫氧环蛋白（Trx）融合表达后与Δα片段进行酶学互补活性分析。结果克隆的α片段为β-galN端1-56多肽片段,定点突变得R14K、R53K和R14K/R53K等3种突变体。融合表达产物经亲和层析后纯度达到90%以上,且4种α片段的纯度基本一致。各纯化产物显示了不同的酶学互补活性,其中R53K突变体的活性远高于R14K和R14K/R53K的,且与野生型α片段的基本一致。结论α片段R53K突变体具有作为CEDIA系统含内部标记位点供体的潜力。

Objective To introduce an internal ε-NH2 for conjugated analyte into donor of cloned enzyme donor immunoassay （ CEDIA ）.Methods α gene of β-galactosidase was cloned from Escherichia coli,and introduced lysine code by site-specific mutagenesis. α genes were fused to Trx tag and expressed under the control of promoter T7 in E. coli Origami （DE3）,then screened by complementation assay with Δα fragment after purification.Results An αgene coding 1-56 aa was cloned,then three mutants R14K、R53K and R14K/R53K were obtained by changing arginine into lysine. The over-expression products of αgenes were purified to a purity over 90%. Of three mutants,R53K showed the highest activity,which was identical to native α fragment＇s and far higher than other mutants＇.Conclusion Mutant R53K could be potentially used as a donor containing internal ε-NH2 for CEDIA.