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 ampli-fication 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 eu-karyotic expression vector and the expression and distribution of hnRNPA1 protein in cells were detected by real-time fluo-rescence 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 rela-tively 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%),ran-dom 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 pro-tein is an unstable hydrophilic protein,which does not contain transmembrane domains and signal peptides,and the se-condary structure is mainly random coil.pcDNA3.0-hnRNPA1-Flag eukaryotic expression vector was successfully con-structed,and hnRNPA1-Flag protein is mainly expressed in cytoplasm.
pighnRNPA1 genebioinformatics analysisconstruction of expression vector
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