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目的通过基因克隆方法制备鲍曼不动杆菌外膜蛋白W(outer membrane protein W,OmpW),为研究其生物学活性及耐药机制奠定实验基础。方法通过PCR方法扩增鲍曼不动杆菌临床株A1株的外膜蛋白W基因(OmpW),构建原核表达载体pET30a/ompW,采用PCR方法和测序鉴定扩增产物。筛选阳性表达载体后转化至大肠埃希菌表达宿主菌BL21中,IPTG诱导表达重组蛋白并进行纯化。结果 pET30a/ompW原核表达重组载体构建成功,基因测序鉴定,其在原核表达系统中高表达,表达产物分子质量单位约为43ku,与预期值相符。表达产物经8mol/L尿素和Tris-HCl处理纯化,得到高纯度的重组OmpW蛋白。结论成功构建鲍曼不动杆菌OmpW蛋白基因重组原核表达系统,并获得高纯度的重组OmpW蛋白,为研究鲍曼不动杆菌外膜蛋白W的生物学活性以及耐药机制奠定了基础。
Objective To prepare outer membrane protein W (OmpW) of Acinetobacter baumannii by gene cloning method, and lay the experimental foundation for studying its biological activity and drug resistance mechanism. Methods The outer membrane protein W gene (OmpW) of Acinetobacter baumannii strain A1 was amplified by PCR and the prokaryotic expression vector pET30a / ompW was constructed. The amplified products were identified by PCR and sequencing. The positive expression vector was screened and transformed into Escherichia coli BL21. IPTG induced expression of the recombinant protein and purified. Results The prokaryotic expression vector pET30a / ompW was successfully constructed and identified by sequencing. The recombinant plasmid pET30a / ompW was highly expressed in prokaryotic expression system. The molecular mass unit of expressed product was 43ku, which was consistent with the expected value. The expression product was purified by 8mol / L urea and Tris-HCl to obtain high-purity recombinant OmpW protein. Conclusion The recombinant prokaryotic expression system of Acinetobacter baumannii OmpW gene was successfully constructed and the recombinant OmpW protein of high purity was obtained, which laid the foundation for the study on the biological activity and drug resistance mechanism of Acinetobacter baumannii outer membrane protein.