鸭DCN基因编码区扩增、序列分析及其在大肠杆菌中的表达、蛋白纯化

采用RT-PCR方法从鸭腿肌总RNA中扩增了鸭核心蛋白聚糖基因编码区序列,进行序列分析,并将编码鸭核心蛋白聚糖成熟蛋白的核酸片段连入原核表达载体pET-32a(+)中,加入IPTG诱导其表达后,亲和层析纯化表达的蛋白。采用SDS-PAGE检测,用质谱方法对表达的蛋白进行鉴定。研究成功克隆出鸭核心蛋白聚糖基因编码区序列,序列分析表明,鸭DCN编码区序列由1074个碱基组成,编码357个氨基酸,其中信号肽有16个氨基酸。将鸭DCN蛋白划分为9个结构域(LRR1-9),结合人DCN序列进行序列分析推测结构域中LRR4、5与TGF-β可能存在一定关系。SDS-PAGE检测和质谱鉴定结果表明获得了分子量为54.7ku的鸭DCN融合蛋白。

Amplification and Analysis of Duck DCN Gene CDS and Its Expression in Escherichia coil and Protein Purification

DCN coding domain sequence of duck was obtained from the total RNA of adult duck using RT-PCR method.Then the duck DCN mature peptide cDNA was inserted into pET-32a(+) vector and transformed into Escherichia coli Rosseta.And then it was induced by IPTG,the recombinant expression products were detected by SDS-PAGE and confirmed by mass spectrum.The results of sequencing analysis showed that the full duck DCN coding domain sequence was 1 074 bp,encoding for 357 amino acids,and the deduced CDS amino acid sequence contained a 16 amino acids putative signal peptide.The DCN amino acid sequence of duck was composed of nine domains,According to the report about human,there might be certain relationship between LRR4、5 and TGF-β.The recombinant expression products were purified by affinity chromatography.The molecular weight of DCN protein was 54.7 ku through SDS-PAGE and mass spectroscopy.