鲍曼不动杆菌外膜蛋白A的克隆表达及纯化

Cloning expression and purification of Acinetobacter baumannii outer membrane protein A

目的:通过分子克隆方法制备鲍曼不动杆菌外膜蛋白A(OmpA)纯化蛋白,为进一步研究鲍曼不动杆菌OmpA蛋白的生物学活性提供基础.方法:首先采用PCR方法扩增鲍曼不动杆菌标准菌株ATCC19606株外膜蛋白A编码基因(OmpA),构建其原核表达载体pET30a/ompA,所得产物经PCR方法和测序进行鉴定.之后筛选阳性表达载体,将其转化至大肠杆菌表达宿主菌BL21,最后将表达的蛋白质进行纯化.结果:重组表达载体pET30a/ompA构建成功,并经PCR方法和测序方法进行鉴定;重组蛋白在所构建的原核表达系统中实现了高表达,纯化后得到了高纯度的OmpA蛋白.结论:本次试验通过分子克隆技术,使鲍曼不动杆菌OmpA蛋白在构建的原核表达系统中成功表达,并且获得了高纯度的目标蛋白,为进一步研究鲍曼不动杆菌外膜蛋白A的生物学活性及抗体的保护作用奠定了基础.

AIM： To provide a basis study for the biological activity of Acinetobacter baumannii outer membrane protein A （ Om- pA） in Acinetobacter baumannii by preparing purified OmpA with molecular cloning technology. METHODS： PCR method was used to amplify coded gene of 0mpA of standard strains ATCC19606, and construct the prokaryotic expression vector pET30a/ompA. The roducts were verified by PCR method. Then the positively expressed vector was selected and was transformed into Escherichia coli expression strain （BL21 strain）. The expression products were purified finally. RESULTS： pET30a/om- pA recombination expression vector was successfully constructed and verified by PCR method. The target protein was highly ex- pressed in prokaryotic expression system, and the ideal OmpA of Acinetobacter baumannii was obtained. CONCLUSION： In this experiment, Aeinetobaeter baumannii outer membrane protein A gene was successfully expressed in the constructed prokaryotic ex- pression system by molecular cloning technology and the highly purified target protein,which provide a basis for further investiga- ting the biological activities of Acinetobacter baumannii OmpA protein and the protective effects of its polyelonal antibody.