目的：克隆和表达人颗粒酶A（ Granzyme A，GzmA）基因的活性片段（ active Granzyme A，aGzmA）并进行活性鉴定。方法：以全长人GzmA基因为模板，PCR扩增aGzmA基因片段，限制性内切酶NdeⅠ和XhoⅠ双酶切后插入原核表达载体pET24a（＋），将重组质粒转化大肠杆菌BL21（DE3），IPTG诱导表达，表达产物经SDS－PAGE和Western blot进行分析。重组蛋白用镍层析柱亲和层析进行纯化后，利用BLT底物溶液进行活性鉴定。结果：PCR扩增得到长约700 bp的基因片段。重组质粒pET24a－aGzmA经酶切和测序证实aGzmA基因序列正确插入载体质粒中。 SDS－PAGE显示在26 kD处有一特异性蛋白条带。 Western blot证实该蛋白可与小鼠抗His 单克隆抗体发生特异性结合。利用镍柱亲和层析法可从包涵体中纯化得到纯度较高的重组蛋白，且具有较好的酶活性。结论：成功制备了具有生物学活性的人颗粒酶A重组蛋白。%Objective:To clone and express active domain of human granzyme A ( aGzmA ) and detect its biological activity.Methods:Human aGzmA gene was amplified by PCR from the full-length human granzyme A and inserted into prokaryotic ex-pression vector pET24a(+).The constructed recombinant plasmid pET24a-aGzmA was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot.The recombinant protein was purified by the Ni2+affinity column chromatography and the enzyme activity was assayed with BLT substrate.Results: A DNA fragment of 700 bp was amplified by PCR.The recombinant plasmid pET24a-aGzmA identified by enzyme-digesting analysis and sequencing showed that aGzmA gene was inserted into vector plasmid correctly.SDS-PAGE analysis showed that there was a specific protein with a relative molecular mass of about 26 kD.Western blot analysis indicated that the protein could react with mouse anti-His monoclonal antibody specifically.The recombinant protein with high purity could be acquired from the inclusion bodies by the Ni2+ affinity column chromatography and the purified protein had good enzyme activity.Conclusion: The recombinant human granzyme A with good biological activity was prepared successfully.
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