穿透支原体铁蛋白的原核表达、纯化及生物信息学分析

Prokaryotic expression,purification and bioinformatics analysis of Ferritin from Mycoplasma penetrans

[目的]表达纯化穿透支原体(Mycoplasma penetrans)铁蛋白(Ferritin)并利用生物信息学方法分析该蛋白的生物学特性。[方法]从NCBI网站上查到Mpe Ferritin的DNA和蛋白序列,并合成Ferritin基因,设计PCR引物,以合成基因为模板扩增Ferritin片段,将其与pET15b载体连接,将连接产物转化到大肠杆菌Top10感受态细胞。挑取阳性菌落,利用PCR和测序验证,提取质粒pET15b-Ferritin并将其转化到大肠杆菌BL21(DE3),用IPTG诱导目的蛋白表达,并使用Ni2+亲和法纯化Ferritin蛋白。然后用生物信息学方法分析Ferritin蛋白的生物学特性。[结果]Mpe Ferritin基因全长为540 bp,编码180个氨基酸。成功构建重组质粒pET15b-Ferritin,表达并纯化得到Ferritin蛋白。Ferritin蛋白定位于细胞质,无信号肽。它的α-螺旋占71.5%,β-转角占6.7%,延伸链占5.0%,无规卷曲占16.8%。由24个同源Ferritin蛋白分子组成24聚体。[结论]通过基因工程方法纯化得到Ferritin蛋白并分析了Ferritin的生物学性质。

[Objective] To prokaryotic express,purify the Mycoplasma penetrans Ferritin protein and analyze its biological properties. [Methods] The sequence of Ferritin protein was searched from the NCBI and the mutatant Ferritin gene was synthesized. The PCR primers were designed,which were used to amplify the Ferritin gene. The DNA fragment of mutatant Ferritin was ligated into pET15 b plasmid and transformed into E. coli Top10. The recombinant plasmid pET15 b-Ferritin was verified by PCR and sequencing,which was extracted from E. coli Top10 and was transformed into E. coli BL21（ DE3）. The target protein was induced by IPTG and purified by using Ni2 ＋affinity. The biological characteristics of Ferrtin were analyzed through bioinformatics methods. [Results]The full-length Mpe Ferritin gene is 540 bp and encodes 180 amino acids. The recombinant plasmid pET15 b-Ferritin was obtained,and the Ferritin was expressed and purified. Ferritin protein locate the cytoplasm and is absent of a signal peptide. It has an α-helix of 71. 5%,a β-turn of 6. 7%,an extended chain of 5. 0% and a random coil of 16. 8%. The tertiary structure of Ferritin protein was a 24-mer which composed of 24 homologous identical subunits. [Conclusion]The full-length Ferritin protein of Mpe was successfully purified using genetic engineering methods and its biological properties was analyzed.