Cloning And Expression Of Cardiac Na/Ca Exchanger Gene Fragment And Acquisition And Functional Research Of Antibodies Against α-1 And α-2 Repeats

Na+-Ca + exchanger(NCX) shares a region of sequence similarity with Na+-K+ ATPase which spans 24 amino acid residues with 46% homology. This Na+-K+ ATPase region in NCX also appears to be involved in the exchanger function, as indicated by site-directed mutagenesis. Thus it is possible that antibody against the Na+-K+ ATPase region may act as the relatively special agonists or antagonists. In this text, NCX gene fragment including the coding region of the Na+-K+ ATPase region was cloned and expressed in Escherichia coli using molecular biology methods. Firstly, NCX gene was amplified from canine brain RNA with RT-PCR. Secondly, this gene fragment was cloned into cloning plasmid pGEM-T Easy to construct recombinant plasmid pGEM-T Easy-NCX(417-997) from which two subclones, pGEM-T Easy-NCXa and pGEM-T Easy-NCXb, were constructed. Then these three recombinants were sequenced which suggested (hat they were the same as the NCX sequence reported previously. Thirdly, the target gene was then inserted into the prokaryotic expression plasmid pET-28b(+). SDS-PAGE analysis of the induced recombinant expression plasmid pET-28b(+)-NCX indicated that the molecular weight of the expression product was about 26kDa, which is in agreement with thededuced molecular weight of this peptide. In conclusion, NCX gene fragment was expressed in Escherichia coli, which contributed to the production of its polyclonal and monoclonal antibodies and laid foundation for further study of NCX.