目的：构建噬菌体β‐GT蛋白的原核重组体系，诱导表达、纯化GST‐β‐GT 融合蛋白并对其进行酶活性测定。方法利用PCR技术从T4噬菌体中扩增β‐GT基因；经T‐A克隆，获得β‐GT基因片段，经酶切连接，构建原核表达载体pGEX‐6P‐1‐β‐GT ；经测序鉴定序列正确后，将所构建的载体转化进入大肠埃希菌BL21（DE3）中进行表达。利用IPTG诱导，经10％ SDS‐PAGE鉴定目的蛋白表达后，采用GST柱纯化目的蛋白；通过酶切和qPCR鉴定其活性。结果成功扩增了噬菌体β‐GT基因；重组质粒在大肠埃希菌BL21（DE3）中诱导表达出GST‐β‐GT融合蛋白；通过GST柱纯化后获得了GST‐β‐GT融合蛋白，纯度达95％；通过酶切和qPCR验证，此GST‐β‐GT融合蛋白具有T4‐β‐GT酶的活性。结论。原核表达GST‐β‐GT融合蛋白具有糖基转移酶活性。%Objective To construct a prokaryotic system to express phageβ‐GT protein fused with GST and puri‐fied the GST‐β‐GT fusion protein .Methodβ‐GT gene was amplified from T4 phage genomic DNA and was cloned into prokaryotic expression vector pGEX‐6P‐1‐β‐GT .After sequencing ,the recombinant plasmid pGEX‐6P‐1‐β‐GT was transformed into Escherichia coli strain BL21 (DE3) .The expression of the target protein was induced by addi‐tion of IPTG to a final concentration of 1 mmol/L .The expressed protein was identified by 10% SDS‐PAGE .The recombinant GST‐β‐GT fusion protein was purified using the GST‐affinity column .Result Theβ‐GT gene was suc‐cessfully amplified from T4 phage genomic DNA and was cloned into vector pGEX‐6P‐1 .The recombinant plasmid was transformed into E .coli strain BL21 (DE3) .The GST‐β‐GT fusion protein was expressed in E .coli strain BL21 (DE3) .After purification with a GST‐affinity column ,pure GST‐β‐GT fusion protein was obtained that has the T4‐β‐GT enzyme activity verified by enzyme digestion and qPCR .Conclusion The recombinant GST‐β‐GT fusion protein has glycosyl transferase activity .
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