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.
ABSTRACT
Objective To explore whether dithiothreitol(DTT)is helpful for PreScission Protease to cut off the GST from GST?CT3 protein. Meth?ods The pGEX?6P?3/CT3 recombinant plasmid was transfected into Escherichia coli BL21,and the GST?CT3 fusion protein was purified by B?PER method. PreScission Protease was applied with 10 mmol/L DTT to cut off the GST,then the SDS?PAGE was performed for identification of the CT3 protein. Results Without DTT,it was very difficult for PreScission Protease to cut off the GST from GST?CT3 protein. However,in the pres?ence of 10 mmol/L DTT,PreScission Protease could cut off the GST easily as identified by SDS?PAGE. Conclusion 10 mmol/L DTT can help Pre?Scission Protease to cut GST from GST?CT3 protein,so as to achieve high concentration of CT3.
ABSTRACT
Objective To clone the coding region and 3′non?coding region of calmodulin 2(CaM2)in guinea pig,to provide the genetic informa?tion for studying the gene function of Calmodulin 2. Methods Total RNA was extracted from heart tissue of guinea pig,the coding region and 3′non?coding region of CaM2 were amplified by RT?PCR and 3′?RACE PCR methods,and the recombinant plasmid was constructed by inserting cDNA of the coding region and 3′non?coding region of CaM2 into the cloning vector by genetic engineering technology followed by DNA sequencing and se?quence analysis. Results The cloned coding region of CaM2 was 450 bp,and the 3′non?coding region of CaM2 was 660 bp. The amino acid se?quences of the coding region of CaM2 was consistent with those of other CaM subtypes,and the 3′non?coding region of CaM2 had low homology with those of other subtypes. Conclusion The cloning of CaM2 coding region and 3′non?coding region in guinea pig was the foundation for further study on the gene function of CaM2 and its role in related diseases.