ABSTRACT
Objective To construct the engineering bacteria expressing the recombinant human Kunitz protease inhibitor domain of amyloid protein precursor variant (rhKD/APPvar)in Pichia pastoris,and to establish the methods suitable for large-scale fermentation and purification of rhKD/APPvar.Methods The rhKD/APPvar expression vector was constructed based on the rhKD/APPvar-pPICZαexpression vector. Two restriction enzyme loci (ApaⅠ and SacⅡ)were added to two flanks of KD/APP and human KD/APP activity center RAM was replaced by the active site of BPTI KAR.After the rhKD/APPvar-pPICZαexpression vector was transformed into Pichiapastoris,optimized expression and purification of rhKD/APPvar was performed.The rhKD/APPvar was purified with cation exchange chromatography and desalting.Results The results of digestion identification and DNA sequencing analysis demonstrated that the recombinant plasmid rhKD/APPvar-pPICZα was successfully constructed and transfected into pastoris X-33. The SDS-PAGE analysis results indicated that rhKD/APPvar expressed after the induction of methanol and the relative molecular weight was 6 700.After a series of experiments the optimal expression conditions of rhKD/APPvar were obtained as follows:the optimal pH was 6.0 and the optimal induction time point was about the 5 th day for the strain.After purified the purity of rhKD/APPvar was about 95%.Conclusion KD/APPvar-pPICZ is successfully constructed;after expression in Pichia pastoris and purification,the rhKD/APPvar protein is obtained.
ABSTRACT
Objective To explore the experience on using duodenoscope to treat hepatolithiasis through normal physiological ways.Methods ERCP,EST were used firstly,then removed calculus of the extrahepatic bile duct.Endoscopic papillary balloon dilatation was used if there was stenosis of bile duct.Removed calculus when it had been crushed in the hepatic duct.Injected decoction to dissolve calculus through endoscopic nasobiliary drainage,or inserted the endoscopic retrograde biliary drainage when the calculus was hard to removded.Results 101 cases of calculus in the extrahepatic bile duct were all removed.215 cases of hepatolithiasis were cleaned out at first time.9 cases were cured 1 week after dissolving calculus through endoscopic nasobiliary drainage.59 cases carried out ERBD,and 21 of them were cured 3 months later,37 patients were still in regular follow-up.33 cases with stenosis of bile duct were treated by endoscopic papillary balloon dilatation,19 of them were cured,the others were inserted with the endoscopic retrograde biliary drainage.19 cases of cholangitic abscess were cured by endoscopic nasobiliary drainage.Conclusion Using duodenoscope to treat hepatolithiasis through normal physiological ways is safe and effectic.