ABSTRACT
Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.
ABSTRACT
Objective To establish an improved hemisection model of spinal cord injury(SCI)in rats with less severe injury and increased sur-vival rate. Methods Female Sprague-Dawley rats(n=20)were randomly divided into 2 groups:standard and improved hemisection model of SCI. Basso-Beattie-Bresnahan(BBB)scores were carried out to determine the hindlimb locomotor function at 1 d,3 d,1 week and 2 weeks post SCI,respectively. In addition,duration of operation,blood loss,location of posterior midline,and success rate of spinal cord exposure during the second surgery were used as the main parameters to evaluate the significance of the new model. Results Compared with the standard hemisection model,the improved hemisection model of SCI exhibited shorter duration of operation,less blood loss,higher BBB score,more accurate location of posterior midline,and higher success rate of spinal cord exposure during the second surgery. Based on the improved hemisection model of SCI, transplantation model of scar tissue in spine cord was then successfully constructed with a success rate above 70%. Conclusion The improved hemisection model of SCI shows significant advantages in many aspects related to the operation,which is applicable to the study of SCI,especially the non-acute SCI.