Bagherpour Ghasem, Fooladi Abbas Ali Imani, Mehrabadi Jalil Fallah, Nourani Mohammad Reza, Einollahi Behzad
Baqiyatallah University of Medical, Sciences Tehran Iran.
Acta Microbiol Immunol Hung. 2011 Dec;58(4):259-71. doi: 10.1556/AMicr.58.2011.4.2.
Uropathogenic Escherichia coli (UPEC) bacteria are the principal cause of urinary tract infections (UTI). Because these bacteria propagate intracellularly, the cellular immune response is an important factor in UTIs. Therefore, we designed a genetic construct to induce a cellular immune response. In order to develop a genetic construct that induces strong cellular immunity against this pathogen, we used the fimH synthetic gene according to mammalian codon usage, and the gene expression was compared with wild type codon usage. Initially, we designed two constructs, pVAX/fimH mam and pVAX/fimH wt, which contain mammalian and wild type codon usage, respectively. The Cos-7 cell line was transfected separately with a complex of pVAX/fimH mam-ExGene 500 poly cationic polymer and pVAX/fimH wt-ExGene 500 poly cationic polymer. Expression of the fimH gene in both constructs in COS7 cells was confirmed by RT-PCR, SDS-PAGE, and Western blotting. Both of the pVAX/fimH cassettes expressed inserted fimH genes (mam and wt) in Cos-7 cells. Our results suggest that codon optimization successfully expressed the fimH gene because the fimH gene with mammalian codon usage is compatible with the eukaryotic expression system. Therefore, mammalian codon usage could be appropriate in a pVAX/fimH construct as a DNA vaccine.
尿路致病性大肠杆菌（UPEC）是尿路感染（UTI）的主要病因。由于这些细菌在细胞内繁殖，细胞免疫反应是尿路感染的一个重要因素。因此，我们设计了一种基因构建体来诱导细胞免疫反应。为了开发一种能诱导针对这种病原体产生强大细胞免疫的基因构建体，我们根据哺乳动物密码子使用情况使用了fimH合成基因，并将该基因表达与野生型密码子使用情况进行了比较。最初，我们设计了两种构建体，即分别包含哺乳动物密码子使用情况和野生型密码子使用情况的pVAX/fimH mam和pVAX/fimH wt。用pVAX/fimH mam-ExGene 500聚阳离子聚合物复合物和pVAX/fimH wt-ExGene 500聚阳离子聚合物复合物分别转染Cos-7细胞系。通过RT-PCR、SDS-PAGE和Western印迹法证实了两种构建体中fimH基因在COS7细胞中的表达。两个pVAX/fimH盒式结构均在Cos-7细胞中表达了插入的fimH基因（mam和wt）。我们的结果表明密码子优化成功表达了fimH基因，因为具有哺乳动物密码子使用情况的fimH基因与真核表达系统兼容。因此，在作为DNA疫苗的pVAX/fimH构建体中，哺乳动物密码子使用情况可能是合适的。
