ABSTRACT
Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
Subject(s)
Humans , Bacterial Proteins , Diphtheria Toxin/genetics , Escherichia coli/genetics , Molecular Chaperones/genetics , Recombinant Proteins/geneticsABSTRACT
Objective Study of the effects of hepatocyte growth factor on inhibit Intimal hyperplasia of the anastomotic stoma after carotid artery bypass grafting.Methods Thirty-two New Zealand white rabbits were randomly divided into control group and the experimental group.The veins were pretreated with saline solution(control group)only or pretreated with HGF(experimental group ;100ng/ml).The vein grafts were harvested at 14 days,28days after operation,HE Stain and Elastic fibrin Stain,The thickness of Intima and media in the vein grafts,intima-media ratio(I/M) was calculated by computer image analysis system.PCNA Immunohistochemistry was performed.Results The thickness of Intima and media in the vein grafts of control group surpassed experimental group significantly(P ＜0.01).At 14d I/M in the vein grafts of control group (0.81 ± 0.05) surpassed experimental group (0.47 ± 0.05) (P ＜ 0.01),At 28d I/M in the vein grafts of control group(0.73 ± 0.01)surpassed experimental group (0.65 ± 0.01) (P ＜ 0.01).The vascular smooth muscle cell proliferation in experimental group was significantly lower than that in control group (P ＜ 0.01).Conclusion Treatment of veins grafts with HGF can significantly inhibit intimal hyperplasia in a rabbit carotid artery bypass grafting model.