猪DECR1基因cDNA的克隆、序列分析及原核表达研究

Molecular Cloning,Sequence Analysis and Expression in E. coli of DECR1 Gene in Pig

旨在研究猪2,4-dienoyl-CoA reductase1(DECR1)基因的结构,揭示该基因的原核表达规律。试验以山西马身猪的肝脏组织为材料,采用RT-PCR与RACE技术克隆了DECR1基因的cDNA全序列,并将其重组于pET32a+原核表达载体中,经酶切、序列鉴定正确后,重组质粒转化大肠杆菌BL21进行诱导表达。结果表明:猪DECR1基因的cDNA全长2352bp,包括987bp的开放阅读框(ORF),53bp的5′非翻译区(UTR)和1312bp的3′-UTR;编码区(CDS)编码328个氨基酸残基与猪(电子预测序列)、牛、人、猩猩、猴、马、犬、鼠相应序列的同源性分别为99%、88%、88%、87%、87%、87%、87%和83%;SDS-PAGE电泳结果显示,在IPTG诱导4h时,外源蛋白表达效率最高;Western blot检测发现经诱导表达的蛋白产物大小约为35ku,与预测的大小一致。猪DECR1基因的克隆和表达研究,为进一步探究该基因的生物学功能及其分子遗传机制提供了理论基础。

This experiment was conducted to obtain sequence of 2,4-dienoyl-CoA reductase 1（DECR1）,and to study its prokaryotic expression.By Reverse Transcription PCR and Rapid Amplification of cDNA Ends（RACE）,the full-length cDNA of DECR1 was cloned from Mashen pig liver,and inserted into the expression vector pET-32a＋.After confirmation by the sequencing and restriction enzyme analysis,the recombinant plasmid was transformed into E.coli BL21.The results indicated that the cDNA of pig DECR1 contained 2 352 nucleotides,including a 987 bp open reading frame（ORF） flanked by a 53 bp 5′-UTR and a 1 312 bp 3′-UTR.Pig DECR1 CDS encoded 328 amino acid residues,which shared 99%,88%,88%,87%,87%,87%,87% and 83% identity with those of Sus scrofa（predicted）,Bos taurus,Homo sapiens,Macaca mulatta,Pan troglodytes,Equus caballus,Canis and Mus musculus,respectively.SDS-PAGE results showed that the recombinant protein was expressed and then the expression level reached the highest level after induced for four hours.Western blot analysis indicated that the molecular weight of the expressed protein was the same as predicted size of approximately 35 ku.Collectively these data provided the important information for further studying the physiological functions and molecular mechanism of pig DECR1 gene.