NDM-1基因的克隆表达及生物信息学分析

Cloning,expression and bioinformatics of NDM-1 gene

目的克隆鲍曼不动杆菌blaNDM-1基因,对NDM-1进行生物信息学分析,表达并纯化重组NDM-1蛋白。方法 PCR扩增鲍曼不动杆菌NDM-1的blaNDM-1基因,克隆至pMAL-p2X质粒,构建重组原核表达质粒pMAL-p2X∶∶NDM-1,对NDM-1进行生物信息学分析。将重组表达质粒转化大肠杆菌Tb1,IPTG诱导表达,经亲和层析纯化重组蛋白MBP-NDM-1。结果重组表达质粒pMAL-p2X∶∶NDM-1经双酶切和测序证实构建正确;生物信息学分析表明,NDM-1的相对分子质量为28 487.86,等电点为5.89,1～28氨基酸为NDM-1的信号肽序列,与其他亚型金属β-内酰胺酶同源性很低;表达的重组蛋白相对分子质量约73 000,主要以可溶形式表达;纯化后的重组蛋白浓度为1.72 mg/ml。结论表达并纯化了可溶性重组蛋白MBP-NDM-1,为后续NDM-1活性功能及其抑制剂的研究奠定了基础。

Objective To clone the blame1 gene of Acinetobacter baumannii, analyze the bioinformatics of NDM-1 gene and protein, then express and purify recombinant NDM-1 protein. Methods The blanam gene of A. baumannii was amplified by PCR and cloned into plasmid pMAL-p2X to construct recombinant plasmid pMAL-p2X ： ： NDM-1, based on which the bioinformatics of NDM-1 gene and protein were analyzed. The constructed recombinant plasmid was transformed to E. coli Tbl and induced with IPTG, and the expressed recombinant protein MBP-NDM-1 was purified by affinity chromatography. Results Restriction analysis and sequencing proved that recombinant plasmid pMAL-p2X ： ： NDM-1 was constructed correctly. Bioinformatic analysis showed that the relative molecular mass and isoelectric point of NDM-1 were 28 487.86 and 5.89 respectively, while the signal peptide sequence was located in amino acids 1 - 28, and NDM-1 was low homologous to the metal 13 lactamase of other subtypes. The expressed recombinant protein, with a relative molecular mass of about 73 000, mainly existed in a soluble form, and reached a protein concentration of 1.72 mg/ml after purification. Conclusion Soluble recombinant protein MBP-NDM-1 was expressed and purified, which laid a foundation of further study on activity, function and inhibitor of NDM-1.