摘要
目的为获得中华大蟾蜍甾醇-4α-羧酸酯-3-脱氢酶(NSDHL)以便于研究其功能,本文建立了该重组蛋白原核表达方法。方法以中华大蟾蜍耳后腺总RNA为模板,采用RT-PCR技术克隆中华大蟾蜍NSDHL基因(Bbg-NSDHL),对Bbg-NSDHL基因进行生物信息学分析,并以大肠埃希菌为宿主菌,采用pCOLD-TF表达载体,构建Bbg-NSDHL原核表达系统,优化诱导表达条件。结果Bbg-NSDHL cDNA全长1521 bp,开放阅读框长1038 bp,编码345个氨基酸,所表达蛋白分子量为38.6 kD,理论等电点为8.70,分子式为C_(1756)H_(2729)N_(455)O_(495)S_(14)。Bbg-NSDHL具有核苷二磷酸糖差向异构酶结构域和短链脱氢酶/还原酶超家族结构域,在270-292处存在1个明显的跨膜区。Bbg-NSDHL单体由5个α-螺旋和9个β-折叠组成。系统进化分析表明,Bbg-NSDHL与两栖动物的NSDHL聚为一类,与哺乳动物遗传距离较远。Rosetta菌株为Bbg-NSDHL重组蛋白表达适合宿主,最佳诱导条件为15℃条件下,细菌生长至OD_(600)值为1.0时,加入0.1 mmol/L的IPTG诱导24 h。去除Bbg-NSDHL跨膜区截短表达可显著提升Bbg-NSDHL重组蛋白表达量。结论本研究首次获得了中华大蟾蜍NSDHL cDNA全长序列,并建立了Bbg-NSDHL原核表达系统,为进一步研究该基因在中华大蟾蜍蟾毒配基类成分生物合成中的作用奠定了基础。
Objective To obtain sterol-4-alpha-carboxylate 3-dehydrogenase(NSDHL)from Bufo bufo gargarizans in order to study its function,a prokaryotic expression method for the recombinant NSDHL of Bufo bufo gargarizans was established in this study.Methods The NSDHL gene of Bufo bufo gargarizans(Bbg-NSDHL)was cloned by RT-PCR,in which the total RNA from the parotid gland of Bufo bufo gargarizans was used as template.E.coli was used as the host bacteria,the Bbg-NSDHL prokaryotic expression system was constructed with pCold-TF expression vector,and the induction and expression conditions were optimized.Results The Bbg-NSDHL cDNA was 1521 bp in length with an open reading frame of 1038 bp,encoded 345 amino acids,and the expressed protein had a molecular weight of 38.6 kD with molecular formula of C_(1756)H_(2729)N_(455)O_(495)S_(14),and a theoretical isoelectric point of 8.70.Bbg-NSDHL has a nucleoside diphosphate sugar epimerase domain and a short chain dehydrogenase/reductase superfamily domain,with 1 distinct transmembrane region at site 270-292.The Bbg-NSDHL monomer consists of fiveα-helix and nineβ-strand.Phylogenetic analysis showed that Bbg-NSDHL clustered with NSDHL from amphibians,but genetically far away from mammals.Rosetta strain was a suitable host for the expression of Bbg-NSDHL recombinant protein.The optimal induction condition was that when the bacteria grow to OD_(600) value of 1.0,0.1 mmol/L IPTG was added for induction for 24 hours at 15℃.Removing the Bbg-NSDHL transmembrane region for truncated expression significantly increased the amount of recombinant protein expressed.Conclusion The full-length NSDHL cDNA sequence of Bufo bufo gargarizans is reported for the first time,and the establishment of the prokaryotic expression system of the Bbg-NSDHL will provide a foundation for further investigation of the biosynthesis of bufadienolides in Bufo bufo gargarizans.
作者
陈曦
武梦云
王东
CHEN Xi;WU Mengyun;WANG Dong(Department of Outpatient,Luoyang Institute of Electro-Optical Equipment of AVIC,Henan Province,Luoyang 417000,China;School of Traditional Chinese Materia Medica,Shenyang Pharmaceutical University,Liaoning Province,Benxi 117004,China)
出处
《中国当代医药》
CAS
2023年第33期21-26,共6页
China Modern Medicine
