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[Construction and expression of diabody [CD3 x Pgp] without Etag]
Juan-Ni Liu1, Ying-Dai Gao, Jin-Hong Wang
1State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academic of Medical Sciences and Peking Union Medical College, Tianjin, China. liujuanni@163.com
Aim:
To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity.
Methods:
In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity.
Results:
The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively.
Conclusion:
The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.
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