[Expression of fusion protein encoding EGFP-EGF1 of rat coagulation factor VII and its binding function].
This study was aimed at clarification of the function of EGF(1) segment in rat coagulation factor VII with tissue factor (TF) by means of the expression of the fusion protein of EGFP-EGF(1). The DNA fragment encoding EGF(1) was amplified from a rat liver tissue by RT-PCR, and then inserted in an EGFP-procaryotic expression vector to construct the recombinant plasmid pET28a-EGFP-EGF(1) which was introduced into the competent cells of E.coli BL21, then an engineering bacteria strain was obtained which was induced by IPTG to express the fusion protein of EGFP-EGF(1). The fusion protein was purified by chromatography on Ni column, and then acted on the rat hemangioendotheliocytes stimulated with LPS to express TF; the binding of the fusion protein to the hemangioendotheliocytes was detected by means of fluorescence microscopy and flow cytometry. The results indicated that EGFP-EGF(1) was highly expressed in the engineering E.coli strain, and successfully purified, and its molecular mass was confirmed as 36 kD by SDS-PAGE. Fluorescence microscopy and flow cytometry had shown that this fusion protein can bind with the TF on the hemangioendotheliocytes. It is concluded that the EGF(1) region of rat coagulation factor can mediate the specific binding of FVII with TF, so as to lay partly the basis for molecular targeting anti-thrombotic therapy.
May
Heng
H
Institute of Hematology, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, Hubei province, China.
Hu
Yu
Y
Zhang
Ying-Hong
YH
Wang
Hua-Fang
HF
Fang
Jun
J
Guo
Tao
T
Wei
Wen-Ning
WN
D000818:Animals; D042783:Endothelial Cells / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D005167:Factor VII; D013925:Thromboplastin