鲍曼不动杆菌UspA蛋白的克隆表达及生物信息学分析

Cloning,expression.purification.and bioinformatic analysis of the UspA protein from Acinetobacter baumannii

目的克隆表达及纯化鲍曼不动杆菌(Acinetobacter baumannii)普遍应急蛋白A(Universal stress protein A.UspA).并进行生物学预测和分析。方法查询鲍曼不动杆菌UspA基因序列,设计特异性PCR引物,以提取的鲍曼不动杆菌基因组DNA为模板,PCR扩增UspA片段。回收目的片段,连接到质粒pET28a.转化大肠埃希菌.37℃培养后,进行PCR扩增并测序。将重组质粒转入表达菌,加人IPTG诱导重组UspA蛋白表达.并经Ni2+亲和层析纯化。采用生物信息学软件分析UspA蛋白的生物学特性。结果获得全长为444 bp的鲍曼不动杆菌UspA基因,且成功构建了重组质粒pET28a-UspA.得到纯度较高的相对分子质量17X103的重组UspA蛋白。该蛋自为细菌的胞质蛋白,其a-螺旋占51%,延伸链占27%,无规卷曲的含量占31%。由2个同源UspA蛋白分子构成二聚体。结论成功克隆表达了鲍曼不动杆菌UspA蛋白。该蛋白可能含B细胞表位,而具有免疫原性,为其生物学活性及耐药机制研究提供了理论基础。

Objective To elone,express.and purify Acinetobucter ba umannii universal stress protein A(UspA)and analyze its biological characteristics.Methods Speific PCR primers were designed in accordance with the sequence of the A.bumanni UspA gene.which was determined by searching the NCBI database.The UspA gene was amplified u-sing the genomic DNA of A.baumanmnii as a template.The target fragment was recovered and ligated into the plasmid pET28a to produce the recombinant plasmid pET28a-Usp and then transformed into E.coli.E.coli was cultured at 37℃.and the recombinant plasmid was verified using PCR and DNA sequencing.The recombinant plasmid was transformed into the expression bacterium.Expression of the UspA protein was induced with IPTG,and the protein was purified u-sing Ni+affinity chromatography.The biological characteristics of the UspA protein were analyzed using different bioin-formatic software.Results The A.baumannii UspA gene.444 bp in length.was obtained via PCR and the recombi-nant plasmid pET28a-UspA was sccessully constructed.A highly pure recombinant UspA protein with a high molecu-lar weight of 17 ku was sucessfully obtained.The UspA protein is a bacterial cytoplasmic protein.Its structure consists of a-helices(51%),extended chains(27%),and random coils(31%)。The tertiary structureof the Usp protein is a di-mer consisting of two homologous UapA protein molecules.Conclusion Recombinant A.baumannii UspA protein was successfully cloned and purified.It may contain B-cell epitopes,and it has strong immunogenicity.These findings pro-vide a theoretical basis for its biological activity and its mechanism of drug resistance.