[Prokaryotic expression, purification and refolding of extracellular ligand binding domains of chick Tie-2 and its immunogenicity].
OBJECTIVE
To study the prokaryotic expression of extracellular ligand binding domains of chick Tie-2, the purification and refolding conditions of the recombinant protein, and to observe its immunogenicity in mouse.


METHODS
A DNA fragment encoding extracellular ligand binding domains of chick Tie-2 was obtained by PCR from a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector PQE30, and was expressed in E. coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected into mouse subcutaneously, and the antiserum of the mouse was analyzed by ELISA and Western blot analysis.


RESULTS
The recombinant protein was highly expressed in E. coli XL-1 blue, and in mouse it produced the antibody which could specifically recognize the recombinant protein.


CONCLUSIONS
The protein of extracellular ligand binding domains of chick Tie-2 can be highly expressed in prokaryotic expression system, and the expressed protein can induce immune response in mouse. These findings are very important for the further study of this protein in anti-angiogenesis and immunotherapy research.
Luo
Yan
Y
Key Laboratory of Biotherapy of Human Diseases of Ministry of Education, Cancer Center, West China Hospital, Sichuan University, Chengdu, China.
Tian
Ling
L
Wu
Yang
Y
Li
Qiu
Q
Liu
Ji-yan
JY
Hu
Bing
B
Su
Jing-mei
JM
He
Qiu-ming
QM
Liu
Cheng-wen
CW
Wei
Yu-quan
YQ
Wen
Yan-jun
YJ
D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D009389:Neovascularization, Pathologic; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D042787:Receptor, TIE-2 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*
D008024:Ligands; D011993:Recombinant Fusion Proteins; D042787:Receptor, TIE-2