文献类型：期刊文献

中文题名：体外拼装苦瓜降糖多肽P基因

英文题名：Assembly of Momordica charantia P-polypeptide Gene In Vitro

作者：张文姬[1];王碧莲[1];郭茸恺[1];赵健[1];王富军[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2011

卷号：24

期号：1

起止页码：94

中文期刊名：中国生物制品学杂志

外文期刊名：Chinese Journal of Biologicals

收录：CSTPCD;;Scopus;CSCD:【CSCD2011_2012】；

基金：国家十一五支撑计划(2006BAI06A162007-2009)

语种：中文

中文关键词：基因拼装;苦瓜降糖多肽P;聚合酶链反应;密码子偏爱性

外文关键词：Gene assembly; Momordica charantia P-polypeptide; Polymerase chain reaction; Codon preference

摘要：目的 以苦瓜降糖多肽P基因为例,探讨用寡核苷酸片段体外拼装基因的方法。方法根据已知苦瓜降糖多肽P的蛋白序列和大肠杆菌的密码子偏爱性,反向翻译出可在大肠杆菌内表达的苦瓜降糖多肽P基因,设计24条40 bp的降糖多肽P基因寡核苷酸片段引物,通过PCR进行寡核苷酸片段的扩增和拼装。将拼装好的多肽P基因的cDNA与pET-32a载体连接后,转化E.coli BL21(DE3),IPTG诱导表达,表达产物经Ni2+-NTA纯化后检测其对四氧嘧啶引起的糖尿病小鼠的降糖活性。结果苦瓜降糖多肽P基因经2轮PCR扩增后,即可见目的 条带;经第3轮PCR扩增,即可见清晰的目的 条带。经测序鉴定,与设计的降糖多肽P基因序列完全一致。重组蛋白在E.coli BL21(DE3)中获得可溶性表达,且可与鼠抗His单抗发生特异性反应。纯化的融合蛋白纯度达90%以上,产量为10 mg/L,其对四氧嘧啶引起的糖尿病小鼠具有降糖活性。结论体外基因拼装法是一种有效获取目的 基因的方法,可进行密码子偏爱性设计,在宿主系统中有效表达目的 基因。
Objective To investigate the method for in vitro gene assembly using oligonucleotide（oligos）using Momordica charantia P-polypeptide gene as an example.Methods The Momordica charantia P-polypeptide gene which might be expressed in E.coli was reversely translated according to the known protein sequence of the said gene and the codon-preference of E.coli,based on which 24 primers for oligos fragments of Momordica charantia P-polypeptide gene,each at a length of 40 bp,were designed for amplification and assembly of oligos by PCR.The assembled cDNA of Momordica charantia P-polypeptide gene was inserted into vector pET-32a,and the constructed recombinant plasmid was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was purified by Ni2＋-NTA and determined for glucose-decreasing activity in alloxan-induced diabetic mice.Results A faint target band was observed after two cycles of PCR amplification of Momordica charantia P-polypeptide gene.However,after the 3rd cycle of PCR amplification,a clear target band was observed,of which the sequencing result was completely consistent with that of designed gene.Recombinant Momordica charantia P-polypeptide was expressed in a soluble form in E.coli BL21（DE3）,which showed specific reaction with mouse anti-His McAb.The purified fusion protein reached a purity of more than 90% and a yield of 10 mg / L,which showed glucose-decreasing activity in alloxan-induced diabetic mice.Conclusion In vitro assembly is an effective approach for synthesis of target genes,which allows the design of codon preference to express effectively in host system.