Cloning of porcine hnRNPA1 gene and construction of its eukaryotic expression vector

【Objective】In this study，the coding sequence（CDS）of the porcine heterogeneous ribonucleoprotein A1 （hnRNPA1）gene was cloned，the eukaryotic expression vector was constructed in order to investigate the structure and biological characteristics of porcine hnRNPA1 protein.【Method】Total RNA of PK-15 cells was extracted by total RNA extraction kit and cDNA was synthesized by reverse transcription. Using this as a template，hnRNPA1 gene specific amplification primers were designed using SnapeGene，and the CDS sequence of hnRNPA1 gene was amplified by PCR. The physicochemical properties，structure and phosphorylation site of hnRNPA1 protein were predicted by ProtParam， ExPASy-ProtScale，TMHMM-1.0，SignalP-6.0，SPOMA，SWISS-MODEL and NetPhos 3.1. pcDNA3.0-hnRNPA1^-Flag eukaryotic expression vector was constructed and transfected into HEK-293T cells. The construction of hnRNPA1 gene eukaryotic expression vector and the expression and distribution of hnRNPA1 protein in cells were detected by real-time fluorescence quantitative PCR，Western blotting and immunofluorescence staining.【Result】The coding sequence of porcine hnRNPA1 gene was successfully cloned. The full length of hnRNPA1 gene CDS sequence was 963 bp，encoding 320 amino acid residues with a molecular weight of 35 kD in hnRNPA1 protein. The theoretical isoelectric point（pI）was 9.27，fat coefficient was 38.34 and instability coefficient was 43.87（greater than 40.00）. The protein structure was relatively unstable and the total average hydrophilic index（GRAVY）was -0.879. It belonged to hydrophilic protein，which did not contain transmembrane domain and signal peptide. The secondary structure consisted of α-helix（16.56%），random coil（72.50%）and extended chain（10.94%），with 48 potential phosphorylation sites. Western blotting results showed that the eukaryotic expression vector pcDNA3.0-hnRNPA1-Flag could successfully express hnRNPA1 protein after transfection into HEK-293T cells. Immunofluorescence staining showed that hnRNPA1 protein was mainly expressed in the cytoplasm.【Conclusion】The CDS sequence of porcine hnRNPA1 gene is successfully cloned. Porcine hnRNPA1 protein is an unstable hydrophilic protein，which does not contain transmembrane domains and signal peptides，and the secondary structure is mainly random coil. pcDNA3.0-hnRNPA1^-Flag eukaryotic expression vector was successfully constructed，and hnRNPA1-Flag protein is mainly expressed in cytoplasm.