鲍曼不动杆菌Omp33-36外膜蛋白片段(20-299aa)的表达与纯化  

Expression and purification of an Acinetobacter baumannii Omp33-36 outer membrane protein fragment(20-299aa)

作　　者：王慧璇 蔡伟 程建军 王胜军[1] 许化溪[1] WANG Hui-xuan;CAI Wei;CHENG Jiang jun;WANG Sheng-jun;XU Hua-xi(Department of Im-munology Laboratory,Institute of Laboratory Medicine,Jiangsu University,Jiangsu,Zhenjiang 212013,China 212013)

机构地区：[1]江苏大学检验医学研究所免疫研究室,江苏镇江212013

出　　处：《中国病原生物学杂志》2020年第8期887-890,896,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81771756)。

摘　　要：目的利用pET-28a蛋白表达载体,在大肠埃希菌BL21(DE3)pLysS中重组表达鲍曼不动杆菌Omp33-36外膜蛋白片段(20-299aa)。方法 PCR扩增获得鲍曼不动杆菌Omp33-36外膜蛋白片段(20-299aa)基因,克隆入pET-28a表达载体,构建重组表达质粒pET28a/Omp33-36aa20-299,转化入大肠埃希菌BL21(DE3)pLysS中,经IPTG诱导蛋白表达,对包涵体表达的蛋白进行变性复性处理,用镍柱亲和层析纯化后进行SDS-PAGE电泳分析。结果PCR扩增获得大小约862bp的目的片段,构建的重组表达质粒pET28a/Omp33-36aa20-299,经双酶切鉴定成功插入目的基因片段,重组质粒转化大肠埃希菌BL21(DE3)pLysS,经IPTG诱导成功表达Omp33-36aa20-299 6His重组蛋白,SDS-PAGE电泳显示重组蛋白分子质量单位约为31.5ku,经镍亲和纯化得到高纯度的重组Omp33-36。结论成功重组表达鲍曼不动杆菌Omp33-36外膜蛋白片段(20-299aa),为进一步研究其对免疫系统的影响奠定了实验基础。Objective To use the pET-28 aprotein expression vector to recombinantly express an Acinetobacter baumannii Omp33-36 outer membrane protein fragment(20-299 aa)in Escherichia coli BL21(DE3)pLysS. Methods The gene coding for the outer membrane protein Omp33-36 fragment(20-299 aa)of A.baumannii was amplified with PCR and cloned into a pET-28 aexpression vector.The recombinant expression plasmid pET28 a/Omp33-36 aa20-299 was constructed and transformed into E.coli BL21(DE3)pLysS.Expression of the protein was induced with IPTG.The denatured and refolded protein was expressed in inclusion bodied.The renatured protein was purified using Ni column affinity chromatography and analyzed using SDS-PAGE electrophoresis. Results A target fragment of about 862 bp was obtained by amplification with PCR.The recombinant expression plasmid pET28 a/Omp33-36 aa20-299 was successfully constructed.Double-enzyme digestion indicated that the target gene fragment was successfully inserted.The recombinant expression plasmid pET28 a/Omp33-36 aa20-299 was transformed into E.coli BL21(DE3)pLysS,and its expression was induced with IPTG.The Omp33-36 aa20-299 6 His recombinant protein was successfully expressed in E.coli BL21(DE3)pLysS.SDS-PAGE electrophoresis indicated that the relative molecular mass of the recombinant protein was approximately 31.5 ku,and a highly-pure recombinant Omp33-36 was obtained using nickel affinity purification. Conclusion A recombinant A.baumannii Omp33-36 outer membrane protein fragment(20-299 aa)was successfully expressed,laying an experimental foundation for further research on its impact on the immune system.

关 键 词：鲍曼不动杆菌 外膜蛋白Omp33-36 蛋白纯化 

分 类 号：R378[医药卫生—病原生物学]