摘要
利用沙门菌膜插入和分泌蛋白(MisL)能展示外源蛋白于细菌表面的能力,构建血管内皮生长因子受体单链抗体(scFv-KDR)的原核表达载体(pYA3332-misL-scFv-KDR),将scFv-KDR展示于减毒沙门菌表面,获得的KDR沙门菌(KDR-X4450)有助于沙门菌靶向肿瘤和肿瘤血管细胞;利用环氧合酶(COX-2)具有高度的肿瘤组织特异性,构建COX-2启动子介导的GFP真核表达载体(pGL3-basic-hCOX2-GFP)。将真核载体转化展示有scFv-KDR的KDR-X4550得到KDR-GFP沙门菌(KDR-GFP-X4550),KDR-GFP-X4550感染肿瘤细胞,COX-2启动子启动外源基因GFP的表达;SDS-PAGE检测scFv-KDR在沙门菌的表达,细菌计数KDR-X4550进入肿瘤细胞的数量;荧光显微镜观察COX-2启动子启动外源基因表达及KDR-GFP-X4550靶向递呈外源蛋白的能力。结果表明:scFv-KDR能展示在沙门菌表面,展示有KDR单链抗体的沙门菌比野生菌更容易进入肿瘤细胞;与正常细胞相比,COX-2启动子能在肿瘤细胞内启动GFP表达,KDR-GFP-X4550具有靶向递呈GFP的能力。这一研究为重组沙门菌进一步应用研究奠定了基础。
Using the membrane insertion and secretion protein(MisL) of Salmonella can display the exogenous protein on the bacterial surface and construct the prokaryotic expression vector of pYA3332-misL-scFv-KDR. By displaying the single chain antibody of vascular endothelial growth factor receptor(scFv-KDR) on the surface of attenuated Salmonella, we obtained KDR Salmonella(KDR-X4550) which can help the attenuated Salmonella target the tumors and tumor vascular cells. Constructed Cyclooxygenase(COX)-2-GFP eukaryotic expression vector(pGL3-basic-hcox-2-GFP) for the COX-2 had the high tumor tissue specificity. By transforming the eukaryotic vector to the KDR-X4550 Salmonella which display the scFv-KDR, the recombinant attenuated Salmonella(KDR-GFP-X4550) was obtained. Then the recombinant attenuated Salmonella infected the tumor cells, and the COX-2 promoter activated the exogenous gene expressed in the tumor cells. The scFv-KDR expressed in the Salmonella was detected by SDS PAGE, and the bacterial count was used to show the numbers of KDR-X4550 Salmonella entering tumor cells. Fluorescence microscope was used to observe the tumor targeted delivery exogenous protein ability of the KDR-GFP-X4550 Salmonella and the ability of COX-2 promoter activating the expression of exogenous gene GFP in tumor cells. The results showed that scFv-KDR could be displayed on the surface of Salmonella, and Salmonella with single chain antibody of KDR could enter tumor cells more easily than wild bacteria. Compared with normal cells, COX-2 promoter can initiate GFP expression in tumor cells, KDR-GFP-X4550 Salmonella had the tumor-targeted delivery ability. These results will provide a foundation for further application of the recombinant Salmonella.
作者
孔桂美
王继军
黄小丽
陶文华
孙国壮
万丹
KONG Guimei;WANG Jijun;HUANG Xiaoli;TAO Wenhua;SUN Guozhuang;WAN Dan(College of Med icine,Yang zhou University,Yangzhou 225009,China;Key Laboratory of Zoonoses of Jiangsu Province/Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses of Jiangsu Province,Yangzhou 225009,China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2020年第1期16-20,26,共6页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
国家自然科学基金资助项目(81272537)
扬州市社会发展项目(YZ2018071)。
关键词
减毒沙门菌
单链抗体
血管内皮生长因子受体
肿瘤靶向递呈
attenuated typhimurium Salmonella
single-chain antibody
vascular endothelial growth factor receptor
tumor-targeted delivery
