Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli.
Induction of specific immunological unresponsiveness by oral autoantigens such as glutamic acid decarboxylase 65 (GAD65) is termed oral tolerance and may be a potential therapy for autoimmune diabetes. However, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. Mucosal adjuvants such as cholera toxin B subunit (CTB) may lower the level of autoantigens required. Here we describe cloning, expression, purification and identification study of the CTB and triple GAD(531-545) epitopes fusion gene. The fusion gene was ligated via a flexible hinge tetrapeptide and expressed as a soluble protein in Escherichia coli BL21 (DE3) driven by the T7 promoter. We purified the recombination protein from the cell lysate and obtained approximately 2.5mg of CTB-GAD((531-545)3) per liter of culture with greater than 90% purity by a Ni-NTA resin column. The bacteria produced this protein as the pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and GAD65. Further studies revealed that oral administration of bacterial CTB-GAD((531-545)3) fusion protein showed the prominent reduction in pancreatic islet inflammation in non-obese diabetic mice. The results presented here demonstrate that the bacteria bioreactor is an ideal production system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes.
School of Medicine, Ningbo University, 818 Fenghua Road, Ningbo 315211, China. zhgong@yahoo.com
Zhaohui
Z
Gong
Xiang'e
X
Long
Lin
L
Pan
Yanping
Y
Le
Qiong
Q
Liu
Shaomin
S
Wang
Junming
J
Guo
Bingxiu
B
Xiao
Mi
M
Zhou
Disen
D
Mei
D000284:Administration, Oral; D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens; D001483:Base Sequence; D019149:Bioreactors; D002772:Cholera Toxin / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D006651:Histocytochemistry; D007108:Immune Tolerance; D007515:Islets of Langerhans / Q000166:cytology; D051379:Mice; D016688:Mice, Inbred NOD; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*
D001324:Autoantigens; D011993:Recombinant Fusion Proteins; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2