[Construction and expression of diabody [CD3 x Pgp] without Etag].
OBJECTIVE
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.


CONCLUSIONS
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.
Liu
Juan-Ni
JN
State 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
Gao
Ying-Dai
YD
Wang
Jin-Hong
JH
Shao
Xiao-Feng
XF
Fan
Dong-Mei
DM
Ji
Qing
Q
Xiong
Dong-Sheng
DS
Yang
Chun-Zheng
CZ
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000276:immunology*; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D001667:Binding, Competitive; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology; D010455:Peptides
D018435:ATP Binding Cassette Transporter, Subfamily B; D018033:Antibodies, Bispecific; D017252:CD3 Complex; D007135:Immunoglobulin Variable Region; D010455:Peptides