Cloning of cDNA of inhibitory protein of rat myocardium H +-ATPase

AIM and METHODS:A pair of primers were designed and synthesized based on ATPase inhibitory protein cDNA sequences of rat myocardium mitochondrial H +-ATPase. A 400bp cDNA fragment was amplified by reverse transcription-polymerase chain reaction(RT-PCR) using mRNA of rat myocardium as template.IF 1 cDNA was blunted with Klenow enzyme and purified using geneclean kit,the PCR amplified cDNA was inserted into pUC19 at Sma Ⅰ site and the recombinant plasmid DNA was used to transform the competent E.coli DH 5α cells.Recombinant plasmids were picked up and miniprepered. RESULTS:A clone,pIF 1,bearing IF 1 cDNA was identified by restriction analysis and PCR amplification using recombinant pUC19 as template.The IF 1 cDNA in the plasmid was found in right orientation by restriction analysis.