Background To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.Methods Bovine enterokinase catalytic subu...Background To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.Methods Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from the duodenal mucosa of a bovine obtained at a wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His · Tag affinity chromatography, and its bioactivity was analyzed.Results Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DMA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using a Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 211 overnight and shown to have a high autocatalytic cleavage activity.Conclusion The EKL gene from a Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.展开更多
BACKGROUND: Currently adopted diagnostic methods for duodenal-biliary and pancreaticobiliary refluxes carry many flaws, so the incidence of the two refluxes demands further larger sample size studies. This study aimed...BACKGROUND: Currently adopted diagnostic methods for duodenal-biliary and pancreaticobiliary refluxes carry many flaws, so the incidence of the two refluxes demands further larger sample size studies. This study aimed to evaluate Western blotting for the diagnosis of refluxes in biliary diseases. METHODS: An oral radionuclide (99)mTc-DTPA test (radionuclide, RN) was conducted for the observation of duodenal-biliary reflux prior to measuring bile radioactivity and Western blotting for detecting bile enterokinase (EK). Pancreaticobiliary reflux was assessed by biochemical and Western blotting tests for biliary amylase activity and trypsin-1, respectively. In accordance with bile sample origin, our samples were classified into ductal bile and gall bile groups; based on each individual biliary disease, we further classified the ductal bile group into five subgroups, and the gall bile group into four sub-groups. Western blotting was conducted to assess the two refluxes in biliary diseases. RESULTS: Consistencies were noted between EK and RN tests when diagnosing duodenal-biliary reflux (P<0.001). The amylase and trypsin-1 tests also showed consistency in diagnosing pancreaticobiliary reflux (P<0.001). Amylase and lipase levels within gall and ductal bile were strongly correlated (P<0.05). In the common bile duct pigment stone group, the EK and trypsin-1 positive rates were found to be insignificant (P>0.05); in the common bile duct cyst group, the EK positive rate was significantly lower than the trypsin-1 positive rate (P<0.05). CONCLUSIONS: Western blotting can accurately reflect duodenal-biliary and pancreaticobiliary refluxes. EK has greater sensitivity than RN for duodenal-biliary reflux. The majority of biliary amylase and lipase comes from the pancreas in all biliary diseases; pancreaticobiliary reflux is the predominant source in the common bile duct cyst group and duodenal-biliary reflux is responsible for the ductal pigment stone group.展开更多
Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into pl...Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.展开更多
基金This study was supported by a National ' 95 ' Key Project grant (No. 96-C02-01-04).
文摘Background To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.Methods Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from the duodenal mucosa of a bovine obtained at a wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His · Tag affinity chromatography, and its bioactivity was analyzed.Results Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DMA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using a Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 211 overnight and shown to have a high autocatalytic cleavage activity.Conclusion The EKL gene from a Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.
文摘BACKGROUND: Currently adopted diagnostic methods for duodenal-biliary and pancreaticobiliary refluxes carry many flaws, so the incidence of the two refluxes demands further larger sample size studies. This study aimed to evaluate Western blotting for the diagnosis of refluxes in biliary diseases. METHODS: An oral radionuclide (99)mTc-DTPA test (radionuclide, RN) was conducted for the observation of duodenal-biliary reflux prior to measuring bile radioactivity and Western blotting for detecting bile enterokinase (EK). Pancreaticobiliary reflux was assessed by biochemical and Western blotting tests for biliary amylase activity and trypsin-1, respectively. In accordance with bile sample origin, our samples were classified into ductal bile and gall bile groups; based on each individual biliary disease, we further classified the ductal bile group into five subgroups, and the gall bile group into four sub-groups. Western blotting was conducted to assess the two refluxes in biliary diseases. RESULTS: Consistencies were noted between EK and RN tests when diagnosing duodenal-biliary reflux (P<0.001). The amylase and trypsin-1 tests also showed consistency in diagnosing pancreaticobiliary reflux (P<0.001). Amylase and lipase levels within gall and ductal bile were strongly correlated (P<0.05). In the common bile duct pigment stone group, the EK and trypsin-1 positive rates were found to be insignificant (P>0.05); in the common bile duct cyst group, the EK positive rate was significantly lower than the trypsin-1 positive rate (P<0.05). CONCLUSIONS: Western blotting can accurately reflect duodenal-biliary and pancreaticobiliary refluxes. EK has greater sensitivity than RN for duodenal-biliary reflux. The majority of biliary amylase and lipase comes from the pancreas in all biliary diseases; pancreaticobiliary reflux is the predominant source in the common bile duct cyst group and duodenal-biliary reflux is responsible for the ductal pigment stone group.
基金the Natural Science Foundation of China (30300186)the Grant of 863 projects from the Ministry of Science & Technology of China (2002AA223354)
文摘Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.
