Objective:To screen the interaction sites of the postsynaptic densitries(PSD-93) and chemokine CX3CL1. Methods:The whole gene synthesis of bait gene PSD-93(1-852aa) was conducted, and the bait gene CX3CL1(1-395aa), mut genes PSD-93-mut1(1-192aa), PSD-93-mut2(1-420aa), PSD-93-mut3(1-535aa), PSD-93-mut4(1-661aa) and CX3CL1-mut(25-357aa) were amplified by PCR. PSD-93 and CX3CL1 genes were recombined into pSos vector to construct bait plasmid pSos-PSD-93-full length and pSos-CX3CL1-full length. PSD-93-mut1, PSD-93-mut2, PSD-93-mut3, PSD-93-mut4 and CX3CL1-mut genes were recombined into pMyr vector to construct mutant plasmids pMyr-PSD-93-mut1, pMyr-PSD-93-mut2, pMyr-PSD-93-mut3, pMyr-PSD-93-mut4 and pMyr-CX3CL1-mut. After restriction enzyme digestion and sequencing, the plasmids were transformed into yeast cdc25Hα and its expression was determined in yeast. The self-activation and cytoplasmic localization were detected. The interaction between PSD-93 and CX3CL1 protein was screened by CytoTrap yeast two-hybrid system. The interaction site of PSD-93 and CX3CL1 protein was screened by CytoTrap yeast two-hybrid system. Results:The recombinant bait plasmid and mutant plasmid were obtained successfully. In the yeast two-hybrid system, the following positive clones were screened by combining the constructed plasmids, which were pSos-PSD-93-full length+pMyr-CX3CL1-full length, pSos-CX3CL1-full length+pMyr-PSD-93-mut3 and pSos-CX3CL1-full length+pMyr-PSD-93-mut4. Thus the specific amino acid sequence of PSD-93 and CX3CL1 binding was identified. Conclusion:In this experiment, the binding sites of PSD-93 and CX3CL1 are located at the 420-535aa sequence of PSD-93 and the 357-395aa sequence of CX3CL1 respectively.
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