[Cloning of CTB-PROIN fusion gene and its expression in Escherichia coli].
A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.
Chen
Li
L
Life Science School of Ningxia University, Yinchuan 750021, China.
Ouyang
Feng-Xiu
FX
Qian
Bing-Jun
BJ
Ren
Hong
H
Wang
Qiang
Q
Jiang
Qing-Wu
QW
Wang
Yu-Jiong
YJ
Liu
Jing-Bo
JB
Liang
Wan-Qi
WQ
Zhang
Da-Bing
DB
D019677:Artificial Gene Fusion; D002772:Cholera Toxin / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D011384:Proinsulin