鲍曼不动杆菌外膜蛋白34的表达纯化及其生物活性分析

发布时间：2018-01-12 12:08

本文关键词：鲍曼不动杆菌外膜蛋白34的表达纯化及其生物活性分析　出处：《生物技术通报》2017年07期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Preparation of Acinetobacter baumannii ATCC 19606 from Acinetobacter baumannii of Acinetobacter baumannii by Gene cloning. Outer membrance protein 34. Omp34, and its biological structure and apoptosis on HEK293 cells were analyzed. The ATCC19606Omp34 gene of Acinetobacter baumannii was amplified by PCR. It was cloned into the expression vector pET28a() to construct the recombinant plasmid pET28a( pET28a- Omp34) and transformed into Escherichia coli BL21. IPTG induced protein expression was purified by nickel affinity chromatography and Omp34 protein was renatured correctly by using L- arginine and reduced glutathione / oxidized glutathione. Biological information was used to predict the biological structure of Omp34 and the secondary structure of Omp34 was determined by circular dichroism (CD) technique. The apoptotic effect of Omp34 on HEK293 cells was detected by flow cytometry and Western blot. The expression vector pET28a (pET28a- Omp34) was successfully constructed. In prokaryotic expression system, soluble protein was obtained by renaturation. Bioinformatics predicted that Omp34 was 尾 -bucket protein, and circular dichroism (CD _ 2) confirmed that Omp34 structure was 尾 -fold structure. The content of 伪-helix is 12, 尾-fold is 39 and 尾-turn angle is 22%. Omp34 can induce apoptosis of HEK293 cells. Acinetobacter baumannii outer membrane protein 34 was successfully obtained by molecular cloning and its secondary structure was analyzed. It is confirmed that it can induce apoptosis of HEK293 cells, which lays a foundation for further study on the biological function and drug resistance mechanism of Acinetobacter baumannii outer membrane protein 34.
【作者单位】：首都医科大学附属北京友谊医院临床检验中心;
【分类号】：R378
【正文快照】： 鲍曼不动杆菌是一种非发酵的革兰氏阴性的机会致病菌,可以引起呼吸机相关肺炎、败血症、泌尿系感染、脑膜炎等疾病,它可以造成严重的免疫系统损害,是医院获得性感染的重要致病菌[1]。近年来由于抗生素的滥用,鲍曼不动杆菌的抗生素耐药问题已经日渐突出,临床出现大量多耐药菌株

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