Interaction between target peptide and complementary peptide by bimolecular fluorescence complementary technology

ABSTRACT

[Objective] Using the bimolecular fluorescence complementation (BiFC) technology, the present experiment aimed to study the interaction relationship and localization of the target peptide and the complementary peptide based on the porcine epidemic diarrhea virus (PEDV) S protein receptor binding site peptide in living cells, so as to provide the foundation and theoretical support for the further use of the peptide in the detection of porcine epidemic diarrhea virus. [Method] The target peptide was designed according to the physical and chemical characteristics of the target protein, such as the amino acid composition, the type of charge, the ability to form intennolecular hydrogen bonds, the strength of polarity, and hydrophobicity;According to the amino acid composition of the target protein, a complementary peptide that interacted with it in theory was designed, and the target peptide and complementary peptide were predicted and analyzed by using bioinfonnatics tools;The target peptide and complementary peptide were inserted into the pBiFC-VC155 and pBiFC-VN173 vector, which was double digested by the EcoRI/XhoI and NotI/SalI, respectively, verified by enzyme digestion and sequencing, and then transfected into Vero cells to study the interaction between the target peptide and the complementary peptide, and the precise localization of BiFC complex in cells. [Result] Bioinfonnatics analysis showed that the target peptide and complementary peptide had hydrophilic and hydrophobic domains, respectively, and the hydrophilic domains were both positively and negatively charged, which could generate electrostatic attraction. The results of enzyme digestion and sequencing showed that the pBiFC-VC155-target peptide and pBiFC-VNI73-complementary peptide plasmids were successfully constructed;Cell transfection experiments showed that the target peptide and complementary peptide could form BiFC complexes in Vcro cells after co-transfection of recombinant plasmids, indicating that they could interact with each other;Indirect immuttolluorescence assay confirmed that the BiFC complex was mainly distributed in the nucleus. [Conclusion] It was confirmed that the peptide designed based on the PEW/ S protein receptor binding site can interact with each other in living cells, demonstrating the feasibility of the peptide for detection.