FLAG标记的Kir2.3通道的构建、表达及其对Kir2.3通道功能的影响

Objective: Flag tag will be inserted into the upstream of Kir2.3-pGEMHE DNA sequence, which will laid a basis for future research. Methods: The DNA base sequence corresponding to the amino acid sequence of Flag peptide was inserted into the N-terminal of Kir2.3 by PCR. The recombinant plasmid DNA FLAG-Kir2.3-pGEMHE would be constructed. The correct clones were chosen by the method of colony PCR and sent for sequencing. Flag-Kir2.3 will be expressed in the Xenopus oocytes. Consequence of Flag tagging on the Kir2.3 channel protein function will be studied. Immunocytochemistry method will be applied to confirm that the Flag-Kir2.3 has expressed on the membrane of the Xenopus oocytes. Results: After examination by sequencing, we were sure that the Flag tag had been inserted into the N-terminal of Kir2.3-pGEMHE. FLAG-Kir2.3 was expressed in the Xenopus oocytes successfully. Channel currents were recorded by TEVC. Immunocytochemistry experiments showed that the Flag tag was fused with Kir2.3 channel protein successfully. Conclusion: FLAG-Kir2.3-pGEMHE, is constructed by means of PCR successfully. Moreover, the Flag tagged protein has expressed stably and Kir2.3 channel function is not affected. These results laid a basis for future research.