Abstract Neonatal Fc receptor (FcRn) can specifically recognize and transport IgG across the mucosal barrier. In order to determine the interaction between porcine (Sus scrofa) FcRn and IgG Fc segment CH2 domain, the porcine IgG CH2 gene was cloned by the reverse transcription (RT-PCR) and further subcloned into prokaryotic expression vector PEGX-4T-1. The fusion protein was induced by isopropyl β-D-thiogalactoside (IPTG) and purified by glutathione S-transferase (GST) Affinity Column. In addition, IgG CH2 gene was subcloned to eukaryotic expression vector pEGFP-C1 and Co-transfection was carried out to determine whether the IgG CH2 and FcRn co-located in African green monkey (Chlorocebus sabaeus) kidney cells (COS-7). The interaction of IgG CH2 and FcRn was verified by coimmunoprecipitation. Furthermore, the binding ability of IgG CH2 and FcRn under different pH condition was detected by ELISA. The results showed the amplified porcine IgG CH2 gene was 330 bp, encoding 110 amino acids. The relative molecular weight of fusion protein GST-CH2 was 38.4 kD. The GST-CH2 in the induced bacteria existed in the form of soluble protein and inclusion body. The soluble GST-CH2 protein will possess the better ability of combining the FcRn. Laser confocal microscope observation showed that the IgG CH2 and FcRn were located in the cytoplasm and had an aggregation phenomenon. The coimmunoprecipitation showed that the corresponding target bands could be detected in all the coprecipitation strips through labeled protein GFP and Flag, which indicated IgG CH2 could bind FcRn. By ELISA, it was found that the IgG CH2 and FcRn had the binding ability when pH value was 6.0, but they were not combined at pH 7.4, suggesting that the combination of IgG CH2 and FcRn was dependent on pH. In conclusion, the results showed that FcRn and CH2 domain of IgG Fc segment had a co-localization and interaction, their combination was pH-dependent, which provides materials for the construction of Fc-based small-size antibody fusion protein.
XU Fa-Zhi,ZHU Zheng-Xuan,DING Xiao-Ling. Cellular Localization and Interaction Between CH2 Domain of IgG Fc Segment and Porcine (Sus scrofa) FcRn[J]. 农业生物技术学报, 2021, 29(3): 521-528.
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