为了获得可在细胞内表达布鲁氏杆菌外膜蛋白31(OMP31)基因的表达载体和慢病毒,试验从羊布鲁氏杆菌M5-90疫苗株PCR获得OMP31基因,连接至p Zero Back/blunt载体,经双酶切鉴定、测序鉴定正确的基因亚克隆到p HAGE-CMV-MCS-IZs Green表达载体上,双酶切鉴定,构建含正确OMP31基因的表达载体p HAGE-OMP31,与包装质粒ps PAX2和包膜质粒p MD2.G共转染293FT细胞进行慢病毒Lentivirus-OMP31包装;获得的Lentivirus-OMP31感染293FT细胞,利用蛋白质印迹(Western-blot)和免疫荧光(IFS)鉴定OMP31蛋白的表达及其免疫原性;LentivirusOMP31体外感染布鲁氏杆菌的宿主细胞模型单核细胞THP-1和人绒毛膜滋养层细胞JEG-3,Western-blot和IFS鉴定OMP31基因的表达,并利用流式细胞(FCM)检测细胞凋亡情况。结果表明:构建了能够在细胞内表达OMP31基因的真核表达载体p HAGE-OMP31和慢病毒LentivirusOMP31,二者感染细胞后表达的OMP31蛋白可与特异性抗体反应,...
【英文摘要】
To obtain the expression vector and lentivirus expressing Brucella melitensis outer membrane protein( OMP) 31 gene. The OMP31 gene was obtained from M5- 90 vaccine strain of Brucella melitensis by PCR,and then inserted into a vector p Zero Back / blunt. The correct OMP31 gene verified by double enzyme digestion and sequencing,was cloned into a expression vector p HAGE- CMV- MCS- IZs Green to construct a expression vector p HAGE- OMP31,which was co- transfected into the 293 FT cells with packaging plasmid ps...
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