Cloning Of Deletion Mutated N-terminus CDNA Of Myosin Light Chain Kinase

Purposes It is very extensive that the smooth muscle is distributed in carrying , play a very important role in maintaining a lot of physiological functions, but its contraction mechanism is very complicated.The well-established mode for the regulation of smooth muscle contraction is: Ca2+ form a complex with calmodulin(CaM), the complex activates myosin light chain kinase (MLCK) to phosphorylate 20kDa myosin light chain(MLC20) at Ser19 to activate myosin ATPase activity,and the inorganic phosphate resulting from Mg2 +-ATP hydrolyzed by activatied myosin Mg2 +-ATPase binds with cross-bridge to increase its Gibbs free energy, wich initiates cyclic cross-bridge-actin interactions and sliding between thick filaments and thin filaments followed by smooth muscle contraction. However, smooth muscle remains tension at resting cytosolic free Ca2+ concentration and its contraction can be detected in the absence of Ca2+ / CaM, the reason for this phenomenon is unknown well. Myosin light chain kinase of smooth muscle(sm MLCK)plays an important role in regulating smooth muscle contraction as a key enzyme with kinase activity as well as non-kinase activity. In recent years, people have very interisting researching about the contraction of smooth muscle non-calcium dependence adjustment. In order to further study its function and the biology activity change, this article makes use of the deletion mutate and establish a model smooth muscle MLCK partial amino acids sequence flaw, the recombinant of MLCK fragment needs to be cloned and expressed.Methods We design forward primer1 and reverse primer2 based on the sequence of pCold /155, to amplify the gene by polymerase chain reaction(PCR).The PCR amplification be double degested by restriction...