鲍曼不动杆菌外膜蛋白34的表达纯化及其生物活性分析

Expression and Purification of Outer Membrane Protein 34 of Acinetobacter baumannii and Analysis of Its Bioactivity

通过基因克隆方法制备鲍曼不动杆菌ATCC 19606(Acinetobacter baumannii ATCC 19606)外膜蛋白34(Outer membrance protein 34,Omp34),并分析其生物结构及对HEK293细胞的凋亡作用。采用PCR方法扩增鲍曼不动杆菌ATCC19606Omp34基因,并将其克隆到表达载体p ET28a(+)以构建重组质粒pET28a(+)-Omp34,转化大肠杆菌BL21,IPTG诱导蛋白表达并用镍柱亲和层析纯化蛋白,使用L-精氨酸和还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)可使Omp34蛋白正确复性,生物信息预测Omp34生物结构,利用圆二色谱(Circular dichroism CD)技术测定Omp34的二级结构,通过流式细胞术和Western blot检测Omp34对HEK293细胞的凋亡作用。成功构建表达载体p ET28a(+)-Omp34,并在原核表达体系中高效表达,通过复性得到可溶蛋白。生物信息预测Omp34为β桶状蛋白,圆二色谱证实Omp34结构为β折叠结构,其中α-螺旋含量为12%、β-折叠为39%、β-转角为22%、无规则卷曲为27%。复性的Omp34可使HEK293细胞发生凋亡。通过分子克隆技术成功获得了鲍曼不动杆菌外膜蛋白34,并解析出其二级结构,证实其可以导致HEK293细胞凋亡,这为将来进一步研究鲍曼不动杆菌外膜蛋白34的生物学功能及其耐药机制奠定了基础。

The present study is to clone and purify the outer membrane protein 34（Omp34）from Acinetobacter baumannii ATCC19606,and to analyze its biological structure and effects on the apoptosis of HEK293 cells. First,the gene of outer membrane protein 34 of A. baumannii ATCC 19606 was amplified by PCR,it was cloned into the p ET28a（＋）vector to generate the p ET28a（＋）-Omp34 expression vector,and the vector was transformed into Escherichia coli BL21 for expression. Then,the recombinant protein was expressedby IPTG induction and purified by Ni-affinity chromatography. L-arginine and GSH/GSSG were added in the dialysis buffer to facilitate theOmp34 protein correct folding. The biological structure of Omp34 was predicted by bioinformatics. The secondary structure of the Omp34 was detected by circular dichroism（CD）.The apoptosis of HEK293 cells induced by Omp34 were measured by flow cytometry and Westernblot. As results,the p ET28a（＋）-Omp34 recombinant expression vector was successfully constructed,the Omp34 was highly expressed inprokaryotic expression system,and the soluble protein was obtained by refolding. Bioinformatics predicted that Omp34 was beta barrel-shape.CD confirmed that the secondary structure of purified Omp34 was beta sheets,of which the α-helix,β-sheets,and β-turn,and random coilof the Omp34 were 12%,39%,22%,and 27%,respectively. The re-natured Omp34 was able to induce the apoptosis of HEK293 cells. Insummary,the Omp34 of A. baumannii can be expressed by molecular cloning technology. Moreover,its secondary structure was analyzed andit is demonstrated that Omp34 can lead to apoptosis of HEK293 cells. This work provides a basis for further study of the biological functions andantibiotic resistance mechanism of A. baumannii Omp34.