羊布鲁杆菌外膜蛋白19(OMP19)的表达及鉴定

Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19

目的构建可表达羊布鲁杆菌外膜蛋白19(OMP19)的真核表达载体,研究OMP19对宿主细胞凋亡的作用。方法以羊布鲁杆菌M5-90疫苗株基因组为模板,PCR扩增脂化型外膜蛋白19(L-OMP19)与非脂化型外膜蛋白19(U-OMP19)基因,产物连接至p Zero Back/blunt载体;经双酶切鉴定、测序正确的基因亚克隆到用于包装慢病毒的表达载体p HAGE-CMV-MCS-IZsGreen上,双酶切鉴定,构建含正确L-OMP19、U-OMP19基因的真核表达载体p HAGE-L-OMP19与p HAGE-U-OMP19。分别将构建好的载体转染293FT细胞、Huh7.5.1细胞和JEG-3细胞,用Western blot法及免疫荧光技术检测L-OMP19、U-OMP19蛋白的表达,用藻红蛋白标记的膜联素Ⅴ/7-氨基放线菌素D(annexinⅤ-PE/7-AAD)双染色结合流式细胞术检测细胞凋亡情况。结果构建能在细胞内表达L-OMP19和U-OMP19基因的真核表达载体p HAGE-L-OMP19和p HAGE-U-OMP19,二者转染细胞后表达的L-OMP19和U-OMP19蛋白可与特异性抗体反应;L-OMP19和U-OMP19不引起Huh7.5.1细胞凋亡,但可引起JEG-3细胞的大量坏死。结论成功构建可用于慢病毒包装的L-OMP19和U-OMP19真核表达载体,胞内重组表达的脂蛋白OMP19具有良好的抗原性,为研究布鲁杆菌脂蛋白在宿主细胞内作用机制提供实验材料。

Objective To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19（ OMP19）,express them in transfected Huh7. 5. 1 and JEG-3 cells,and analyze their role in cell apoptosis. Methods Brucella melitensis lipidated OMP19（ L-OMP19） gene and unlipidated OMP19（ U-OMP19） gene were amplified by PCR and inserted into the vector p Zero Back / blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaⅠ and Bam HⅠ double digestion and sequencing were cloned into the lentivirus expression vector p HAGE-CMV-MCS-IZs Green to construct vectors p HAGE-L-OMP19 and p HAGE-U-OMP19,which were separately transfected into 293 FT cells,Huh7. 5. 1 and JEG-3 cells.L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE /7-AAD staining was used to detect the cell apoptosis. Results The lentiviral vectors p HAGE-LOMP19 and p HAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death,but did not induce the apoptosis of Huh7. 5. 1 cells. Conclusion The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity,which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.