重组金属硫蛋白HcMT的原核表达及其与金属离子竞争反应

Prokaryotic expression of recombinant metallothionein HcMT and its competitive reaction with metal ions

[目的]在不同p H范围内和不同时间下检测重组Hc MT结合的锌、铜离子的含量,对金属离子竞争结合金属硫蛋白的特征进行分析。[方法]构建重组表达载体,将重组质粒p ET-32a-Hc MT进行原核表达,诱导时加入Zn2+,获得菌体后超声破碎,上清中添加Cu2+,分离纯化融合蛋白Trx A-Hc MT,检测金属硫蛋白溶液紫外吸收情况。样品硝化后用ICP-OES检测金属硫蛋白结合的金属离子含量。[结果]在p H 3~9的范围内,重组Hc MT上结合的Zn2+含量随着p H值升高而升高。加入Cu2+后,重组Hc MT上结合的Cu2+浓度上升,Zn2+浓度下降。在p H 3时,重组Hc MT结合金属离子的比值Cu2+/Zn2+为最小值0.68,在p H 8时Cu2+/Zn2+为最大值1.48,有利于Cu2+竞争。当p H 8时,不同时间下Zn-Hc MT与Cu2+的竞争结果表明1 h时锌铜有竞争高峰。[结论]证明了铜离子可以取代结合在重组HcMT上的锌离子,在p H 7~9范围中结合1 h竞争效率最高,该研究为重组Hc MT在重金属解毒方面的应用奠定基础。

[Objective]The content of zinc and copper ions bound by recombinant HcMT was detected in different pH ranges and at different time,and the characteristics of metal ion competition and metallothionein were analyzed.[Method]The recombinant expression vector was constructed,and the recombinant plasmid pET-32a-HcMT was expressed in prokaryotic expression.Zn2+was added during induction,and the cells were sonicated and disrupted.Cu2+was added to the supernatant to isolate and purify the recombinant protein HcMT.Detect the UV absorption of the metallothionein solution.After the sample was nitrated,the metallothionein-bound metal ion content was detected by ICP-OES.[Result]The bound Zn2+content on the recombinant HcMT in the range of pH 3-9 increased with the increase of pH.After the addition of Cu2+,the concentration of bound Cu2+on the recombinant HcMT increased and the concentration of Zn2+decreased.At pH 3,the ratio of recombinant HcMT to metal ion Cu2+/Zn2+is 0.68,and Cu2+/Zn2+is 1.48 at pH 8,which is useful for Cu2+competition.When pH 8,the competition between Zn-HcMT and Cu2+at different times showed that zinc-copper had a competitive peak at 1 hour.[Conclusion]It was proved that copper ions can replace zinc ions bound to recombinant HcMT,and the competition efficiency is the highest in the range of pH 7-9 for 1 hour.This study lays a foundation for the application of recombinant HcMT in heavy metal detoxification.