Expression,cloning and activity assay of active domain of human granzyme A / 中国免疫学杂志
Chinese Journal of Immunology
;
(12): 77-81, 2015.
Article
in Chinese
| WPRIM
| ID: wpr-458444
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Immunology
Year:
2015
Type:
Article
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