人颗粒酶A活性段的表达、纯化及其活性鉴定

Expression,cloning and activity assay of active domain of human granzyme A

目的:克隆和表达人颗粒酶A(Granzyme A,Gzm A)基因的活性片段(active Granzyme A,a Gzm A)并进行活性鉴定。方法:以全长人Gzm A基因为模板,PCR扩增a Gzm A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p ET24a(+),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p ET24a-a Gzm A经酶切和测序证实a Gzm A基因序列正确插入载体质粒中。SDS-PAGE显示在26 k D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（ a Gzm A） and detect its biological activity. Methods： Human a Gzm A gene was amplified by PCR from the full-length human granzyme A and inserted into prokaryotic expression vector p ET24a（ ＋）. The constructed recombinant plasmid p ET24a-a Gzm A 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 p ET24a-a Gzm A identified by enzyme-digesting analysis and sequencing showed that a Gzm A 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 k D. 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.