【摘要】 目的构建人鸟氨酸脱羧酶抗酶(OAZ)突变基因,重组至原核表达载体中并表达出重组蛋白。方法采用基因定点突变技术在OAZ基因TCCTGATG(199~206)位置造成第202位碱基T碱基缺失,使核糖体的阅读框+1移位,连续表达OAZ基因的ORF2部分。测序鉴定后,重组质粒转化BL21菌,进行突变基因的蛋白表达。结果重组质粒经测序后确认202位碱基T缺失,其余碱基序列无变化。重组的突变基因转入BL21后在IPTG诱导下表达OAZ全长蛋白,SDS-PAGE分析在相对分子质量45900左右出现新的蛋白条带。结论成功构建人OAZ突变基因重组质粒,转化BL21后表达出融合的OAZ全长蛋白,为进一步研究其临床应用打下基础。更多还原

【Abstract】 Objective To clone human ornithine decarboxylase antizyme (OAZ) mutation gene and express its recombinant protein. Methods The mutant site was induced by site-directed mutagenesis.The base T locted at TCCTGATG (199-206) was deleted so that the ORF can +1 frameshift and the ORF2 gene can be expressed consecutively. After being sequenced by Sanger dideoxymediated chain termination method, the recombinant vector was transformed into E.coli BL21 to express the protein induced by IPTG. Results The T base was convinced to have been deleted while the other bases remaining unchanged. OAZ protein was expressed after IPTG induction when the recombinant gene was transferred into E.coli BL21. A new protein band about 45 900 was recognized on SDS-PAGE. Conclusion The successful cloning of OAZ-mutation gene and the expression of its recombinant protein in BL21 provides a base for its application in the development of cancer therapy.更多还原