Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization.
Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization.
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耻垢分枝杆菌的遗传改变可改善分枝杆菌介导的质粒 DNA 转移至哺乳动物细胞和 DNA 免疫。
DOI：
10.1128/iai.01877-06
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发表时间：
2007
影响因子：
3.1
通讯作者：
Fennelly,GlennJ
中科院分区：
文献类型：
--
作者：
Mo,Yongkai;Quanquin,NatalieM;Vecino,WilliamH;Ranganathan,UmaDevi;Tesfa,Lydia;Bourn,William;Derbyshire,KeithM;Letvin,NormanL;JacobsJr,WilliamR;Fennelly,GlennJ
分枝杆菌瞄准并持续存在于吞噬单核细胞内，是强佐剂，使其成为DNA疫苗的有吸引力的候选载体。我们描述了分枝杆菌向哺乳动物细胞传递转基因的能力，以及各种细菌染色体突变对体内和体外转移效率的影响。首先，我们通过显微镜和荧光激活细胞分选分析观察了耻垢分枝杆菌感染吞噬细胞系和非吞噬细胞系后绿色荧光蛋白的表达。bovisBCG在真核启动子的控制下含有编码荧光基因的质粒。接下来，我们比较了使用m的基因转移效率。含有染色体插入或缺失的卡介苗，可引起早期裂解、超偶联或质粒拷贝数增加。我们观察到质粒转移到感染m的真核细胞的水平显著增加（尽管只有1.7倍）。smegmatishyperconjugation mutants.M。过多表达pAL5000复制蛋白（Rep）的smegmatisres菌株，质粒拷贝数增加了10倍，向HeLa细胞和J774细胞的基因转移效率分别增加了3.5倍和3倍。pAL5000是许多分枝杆菌构建体中含有复制子的质粒。虽然过表达Rep的卡介苗株不能恢复，但携带复制突变的质粒的卡介苗将基因转移到J774细胞的数量增加了三倍。此外,M。与野生型对照相比，过表达Rep的smegmatisres菌株在体内增强了基因转移。携带编码人类免疫缺陷病毒gp120的质粒（pgp120hE）的分枝杆菌免疫小鼠后，在支持高拷贝数的菌株中，脾细胞和外周血单核细胞对gp120特异性CD8 t细胞的反应分别提高了两倍（P< 0.05）和三倍（P< 0.001）。这些反应的幅度大约是肌肉注射pgp120hE.M后观察到的一半。耻毛杆菌和其他非致病性分枝杆菌是DNA疫苗递送的有希望的候选载体。
Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines byMycobacterium smegmatisorM. bovisBCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer usingM. smegmatisor BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected withM. smegmatishyperconjugation mutants.M. smegmatisstrains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation inoriMresulted in a threefold increase in gene transfer to J774 cells. Moreover,M. smegmatisstrains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120hE) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P< 0.05) and threefold (P< 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120hE.M. smegmatisand other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.