[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlappe...[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.展开更多
In this case report we present an elderly patient who was referred to our hospital with recurrent episodes of cholangitis that persisted after placement of fi ve metal stents for a distal common bile duct(CBD) stenosi...In this case report we present an elderly patient who was referred to our hospital with recurrent episodes of cholangitis that persisted after placement of fi ve metal stents for a distal common bile duct(CBD) stenosis.All metal stents were endoscopically removed from the CBD by forceps after balloon dilatation of the papilla.A profoundly dilated CBD with sludge and concrements was seen.To ensure adequate bile drainage an enteral metal stent was inserted in the CBD.This case shows that proximally migrated uncovered metal stents in the CBD can be safely removed endoscopically under certain circumstances.We suggest that in the case of a CBD drainage problem due to an extremely dilated CBD,placement of an enteral metal stent in the CBD could be considered,especially in patients who are unfi t for surgery.展开更多
基金Supported by the National High Technology Research and Development Program of China(863Program)(2006AA03Z451)Shanghai Basic Research Key Program(10JC1400300)+1 种基金National Natural Science Foundation of China(31070698)the Fundamental Research Funds for the Central Universities(12D10520)~~
文摘[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.
文摘In this case report we present an elderly patient who was referred to our hospital with recurrent episodes of cholangitis that persisted after placement of fi ve metal stents for a distal common bile duct(CBD) stenosis.All metal stents were endoscopically removed from the CBD by forceps after balloon dilatation of the papilla.A profoundly dilated CBD with sludge and concrements was seen.To ensure adequate bile drainage an enteral metal stent was inserted in the CBD.This case shows that proximally migrated uncovered metal stents in the CBD can be safely removed endoscopically under certain circumstances.We suggest that in the case of a CBD drainage problem due to an extremely dilated CBD,placement of an enteral metal stent in the CBD could be considered,especially in patients who are unfi t for surgery.
