Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.

Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.

Objective To evaluate the roles of PI3K/Akt and JAK/STAT signal transduction pathways in reduction of myocardial ischemia/reperfusion (I/R) injury by postconditioning with α subunit-containing nicotinic acetylcholine receptor (α7nAChR) agonist in rats.Methods Sixty Sprague-Dawley rats,weighing 290-320 g,were randomly divided into 4 groups (n =15 each):I/R group,ischemic preconditioning group (IPC group),ischemic postconditioning group (IPOC group) and postconditioning with specific α7nAChR agonist PNU282987 group ( PNU group ).Myocardial I/R was produced by 30 min occlusion of left anterior descending coronary artery followed by 180 min reperfusion in the 4 groups.The animals were subjected to 3 cycles of 5 min myocardial ischemia and 5 min reperfusion before 30 min myocardial ischemia in IPC group.The animals underwent 3 cycles of 10 s myocardial ischemia at 5 s intervals before 180 min reperfusion in group IPOC.PNU282987 2.4 mg/kg was injected intraperitoneally immediately before the reperfusion.At 60 min of reperfusion,5 rats in each group were sacrificed and the hearts were removed to determine the expression of Akt and STAT3 mRNA,phosphorylated Akt (p-Akt) and phosphorylated STAT3 (p-STAT3) in myocardial tissues.The left 10 rats in each group were sacrificed at 180 min of reperfusion and the hearts were removed to measure the infarct size.Results Compared with I/R group,the expression of STAT3 mRNA and p-Akt was significantly up-regulated in IPC group,and the expression of p-Akt and p-STAT3 was significantly up-regulated in IPOC group ( P ＜ 0.05).The infarct size was significantly reduced in IPC,IPOC and PNU groups compared with I/R group ( P ＜ 0.05 ).Conclusion The mechanism by which α7nAChR agonist postconditioning reduces myocardial I/R injury is not related to PI3K/Akt and JAK/STAT signal transduction pathways in rats.