PurposeIn this study, the CPN10 gene was cloned from Banana mini-pig inbred line (BMI) and its structural and functional properties were preliminary explored from the subcellular, mRNA levels and bioinformatics, which laid the foundation for the research on the role of this gene in immune rejection of pig-human xenotransplantation.
MethodsThe cDNA sequence of BMI CPN10 was obtained by RT-PCR, and the recombinant eukaryotic expression vector pEGFP-C1-CPN10 of target gene and reporter gene EGFP was constructed by molecular cloning technology. Then, the recombinant vector was transfected transiently into porcine kidney cells (PK15) to confirm CPN10 localization in cells by tracing EGFP. Further, mRNA expression characteristics of multi-tissue were examined by qPCR techniques. Finally, structural characteristics of CPN10 protein were analyzed by bioinformatics.
ResultThe CDS sequence of BMI CPN10 was 309 bp with GenBank accession number KM098149. The fusion expression vector pEGFP-C1-CPN10 of green fluorescence protein and CPN10 gene was successfully constructed and transfected into PK15 cells. The target proteins were mainly detected in the cytoplasm by tracing the green fluorescent protein. BMI CPN10 mRNA had the highest expression in the skin, higher expression in the liver and adrenal glands, and was moderate, low or hardly expressed in the remaining tissues. Bioinformatics analysis indicated the CPN10 encoded 102 amino acids with molecular weight of 10.93 ku and isoelectric point of 8.89, containing a conserved CPN10 domain and 5 phosphorylation sites. The secondary structure of the CPN10 protein is mainly extended chain structure without transmembrane helical structure and signal peptide.
ConclusionThe CPN10 gene was located in the cytoplasm and highly expressed in the skin, which provided a theoretical basis for further study on the function of CPN10 gene in rejection of xenotransplantation.
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