Expression,cloning and activity assay of active domain of human granzyme A
10.3969/j.issn.1000-484X.2015.01.016
- VernacularTitle:人颗粒酶A活性段的表达、纯化及其活性鉴定
- Author:
Xuefei HU
;
Wenzheng JIANG
- Publication Type:Journal Article
- Keywords:
Granzyme A;
Cloning;
E.coli;
Expression;
Activity assay
- From:
Chinese Journal of Immunology
2015;(1):77-81
- CountryChina
- Language:Chinese
-
Abstract:
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.
