【摘要】 【目的】克隆猪源异质性核糖核蛋白A1（hnRNPA1）基因编码区（CDS）序列并构建其真核表达载体，为探究猪源hnRNPA1蛋白结构及其生物学特性提供理论参考。【方法】利用总RNA提取试剂盒提取PK-15细胞总RNA并反转录合成cDNA，以此为模板，采用SnapeGene设计hnRNPA1基因特异性扩增引物，PCR扩增获得hnRNPA1基因CDS序列。通过ProtParam、ExPASy-ProtScale、TMHMM-1.0、SignalP-6.0、SPOMA、SWISS-MODEL和NetPhos 3.1等预测hnRNPA1蛋白的理化性质、结构及磷酸化位点。构建pcDNA3.0-hnRNPA1^-Flag真核表达载体并转染HEK-293T细胞，通过实时荧光定量PCR、Western blottiing和免疫荧光染色试验检测hnRNPA1基因真核表达载体构建情况、hnRNPA1蛋白在细胞中的表达及分布情况。【结果】成功克隆猪源hnRNPA1基因CDS序列。生物信息学分析结果显示，hnRNPA1基因CDS序列全长963 bp，编码320个氨基酸残基，hnRNPA1蛋白分子量约为35 kD，理论等电点（pI）为9.27，脂肪系数为38.34，不稳定系数为43.87（大于40.00），蛋白结构相对不稳定，总平均亲水性指数（GRAVY）为-0.879，属于亲水性蛋白，不含跨膜结构域和信号肽，二级结构由α-螺旋（16.56%）、无规则卷曲（72.50%）和延伸链（10.94%）组成，存在48个潜在的磷酸化位点。Western blotting检测结果显示，真核表达载体pcDNA3.0-hnRNPA1^-Flag转染HEK-293T细胞后能成功表达hnRNPA1蛋白。免疫荧光染色结果显示，hnRNPA1蛋白主要在胞质中表达。【结论】成功克隆猪源hnRNPA1基因CDS序列。猪源hnRNPA1蛋白为不稳定的亲水性蛋白，不含跨膜结构域和信号肽，二级结构主要为无规则卷曲。成功构建pcDNA3.0-hnRNPA1^-Flag真核表达载体，hnRNPA1蛋白主要在胞质中表达。更多还原

【Abstract】 【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 320amino acid residues with a molecular weight of 35 kD in hnRNPA1 protein.The theoretical isoelectric point （pI） was9.27,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 secondary 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.更多还原