金黄色葡萄球菌LukS-PV-GFP融合蛋白的载体构建及表达纯化  

Vector construction and expression purification of Staphylococcus aureus LukS-PV-GFP fusion protein

作　　者：汪自然 常文娇 戴媛媛 马筱玲 Wang Ziran;Chang Wenjiao;Dai Yuanyuan(Dept of Clinical Laboratory,The Affiliated Prouincial Hospital of Anhui Medical University,Hefei 230001)

机构地区：[1]安徽医科大学附属省立医院检验科

出　　处：《安徽医科大学学报》2019年第12期1903-1907,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81572065)

摘　　要：目的构建LukS-PV与GFP融合蛋白表达载体,在大肠杆菌中表达纯化并鉴定。方法煮沸法提取金黄色葡萄球菌铜23的DNA作为模板,PCR扩增得到LukS-PV基因产物,经胶回收后与pMDN8T质粒连接过夜后转化至感受态菌DH5$中。提取质粒经PCR、测序鉴定无误,双酶切后连接到peW8a和6HTFP载体上,先转化至感受态菌DH5$再转化到BL21宿主菌中。对LukS-PV-GFP表达产物使用SDS-PAGE电泳分析,并取诱导前和IPTG诱导5 h菌液涂片置于荧光显微镜下观察。使用Western blot对诱导前及纯化后蛋白进行鉴定。结果成功构建了LukS-PV与GFP融合蛋白表达载体,诱导纯化后获得较高浓度和纯度的重组融合蛋白,且诱导前无荧光,诱导后宿主菌BL21可在荧光显微镜下显示绿色荧光。Western blot结果显示纯化后蛋白出现特异性条带&结论成功地纯化得到了具有生物学活性的LukS-PV-GFP重组融合蛋白,为进一步研究LukS-PV的功能定位和相互作用奠定了基础.Objective To construct a fusion protein expression voctor of LukS-PV and GFP,and to purify and i dentify the expression vector in Escherichia coli.Methods The DNA of Staphylococcus aureus Cu 23 was extracted by boiling method as a template,and the LukS-PV gene product was obtained by PCR amplification.After gel re-covery,it was connected with pMD-18T plasmid overmight and transformed into DH5x of the susceptible bacteria.The extracted plasmids were identified as correct by PCR and sequencing.After double enzyme digestion,the plas-mids were connected to pet28a-N6H-GFP vector,which was first converted to the host bacterium DH5a and then to the host bacterium BL21.The expression products of LukS-PV-GFP were analyzed by SDS-PAGE electrophoresis.Before induction and after IPTG induction for 5 h bacterial liquid smear was taken and observed under fluorescence microscope.Western blot was used to identify the pre-induction and post-purification proteins.Results The fusion protein expression vector of LukS-PV and CFP was successfully constructed,and the recombinant fusion protein with relatively high concentration and purity was obtained after induction and purification,without fluorescence be-fore induction,and the host bacterium BL21 could show green fluorescence under fluorescence microscope after in-duction.W estern blot results showed that the purified protein showed speeific bands.Conclusion The recombinant LukS-PV-GFP fusion protein with biological activity is successfully purified.

关 键 词：杀白细胞素 绿色荧光蛋白 原核表达 

分 类 号：R394.3[医药卫生—医学遗传学]