人鸟氨酸脱羧酶抗酶1突变基因的克隆与原核表达

Cloning and Prokaryotic Expression of Mutated Human Ornithine Decarboxylase Antizyme 1

克隆人鸟氨酸脱羧酶抗酶1(Homo sapiensornithine decarboxylase antizyme 1,HOAZ1)开放性阅读框+1核糖体移码位点缺失的突变基因,构建突变基因的原核表达质粒,分离纯化其原核表达的重组蛋白。采用巢式-PCR和重叠延伸-PCR技术,从人非小细胞肺癌细胞株A549的cDNA中获得人类鸟氨酸脱羧酶抗酶1开放性阅读框+1核糖体移码(+1RF)位点缺失突变的基因序列(DM-HOAZ1)。将该序列克隆到原核表达载体pET-28a(+)后,转化表达菌Rosseta(DE3)感受态细胞。阳性克隆用IPTG诱导重组蛋白表达,然后在尿素变性条件下经Ni-NTA树脂亲和层析纯化重组HOAZ1。原核表达和纯化的HOAZ1重组蛋白用Western Blot鉴定。结果显示,成功获得HOAZ1开放阅读框中+1RF位点缺失的突变基因和该突变基因的原核表达质粒pET-28a(+)/DM-HOAZ1;用pET-28a(+)/DM-HOAZ1转化大肠杆菌后,HOAZ-1可被IPTG诱导性高表达,且表达量随诱导时间延长递增;原核表达的HOAZ1可用Ni-NTA树脂亲和层析有效纯化。建立了原核表达和分离纯化HOAZ1蛋白的试验方法,为进一步研究HOAZ1的功能和临床应用奠定了基础。

It was to clone human ornithine decarboxylase antizyme 1 with deleted-mutation in ＋ 1 ribosomal frame shifting（ ＋ 1RF） site（ DM-HOAZ1 ）, and to establish a method for prokaryotic expression and purification of the HOAZ1. Nest-PCR and overlapping extension-PCR were used to amplify DM-HOAZ1 from cDNA of human non-small lung cancer cell line A549. DM-HOAZI was then inserted into pET-28a（ ＋ ）vector to construct plasmid pET-28a （ ＋ ）/DM-HOAZ1 for prokaryotic expression, pET-28a （ ＋ ）/DM- HOAZ1 was transformed into E. coli Rosseta（ DE3 ）. The expression of recombinant protein in host cells was induced by 1.0 mM IPTG and resulted recombinant HOAZ1 protein was purified under denature condition by Ni-NTA affinity chromatography, Western blotting assay was used to detect the expression and purification of recombinant protein. Results indicated that a signal nucleotide in ＋ 1RF site was successfully deleted in DM-HOAZ1 compared to natural ORF of HOAZ1. DM-HOAZ1 was cloned into pET-28a（ ＋ ） vector and then transformed into Rosseta（DE3）. DM-HOAZ1 recombinant protein with 6 x His tag was highly expressed in the host cells by IPTG induction and could be purified effectively by Ni-NTA affinity chromatography. Therefore, it proved that an alternative method for prokaryotic expression and purification of the human OAZ1 was successfully established.