摘要
目的:了解问号钩端螺旋体(简称钩体)fliR基因的致病性。方法:分别从问号钩体黄疸出血群56601株DNA和pET42a质粒中扩增fliR基因和kana基因。构建fliR基因自杀质粒,并设计和合成特异性siRNA。采用分别基于自杀质粒的基因敲除、基于siRNA的基因沉默技术构建问号钩体fliR基因突变株,并用PCR、测序、半定量RT-PCR进行鉴定。采用小鼠单核-巨噬细胞J774A.1黏附试验和流式细胞术,检测敲除fliR基因的问号钩体突变株56601fliR-Kana、siRNA阻断fliR基因的问号钩体突变株56601siRNA-R2株黏附细胞,及诱导细胞坏死或凋亡能力的变化。结果:所克隆的fliR基因与GenBank中相应基因的核苷酸和氨基酸序列相似性分别为99.9%和100%,所克隆的kana基因核苷酸序列与pET42a图谱完全相同。56601fliR-Kana可在含氨苄青霉素和卡那霉素的EMJH培养基中生长,PCR和测序结果证实56601fliR-Kana株fliR基因中插入了kana基因。56601fliR-Kana株fliR基因mRNA水平明显下降(P<0.01),56601siRNA-R2株fliR基因mRNA水平也低于野生株(P<0.05)。56601fliR-Kana和56601siRNA-R2黏附J774A.1细胞的能力基本消失,诱导J774A.1细胞坏死或凋亡的能力也明显减弱(P<0.01)。结论:fliR基因是问号钩体毒力相关基因,其功能与问号钩体黏附细胞、诱导细胞凋亡或坏死密切相关。
Objective： To investigate the pathogenicity of Leptospira interrogans fliR gene to J774A. 1 cells. Methods： fliR gene from L. interrongans serogroup Icterohaemorrhagiae serovar lai strain 56601 and kana gene from plasmid pET42a were amplified by PCR. Suicide plasmid of fliR gene was constructed; and specific siRNA for fliR gene was designed and synthesized, fliR gene mutants were constructed by gene knock-out with suicide plasmid （56601^fliR-Kana） and gene silencing with siRNA （56601^siRNA-R2）. The mutants were identified by PCR,sequencing and semiquantitative RT-PCR. Adhesion to mouse mononuclear macrophage J774A. I and induction of cell necrosis and apoptosis by 56601^fliR-Kana and 56601^siRNA-R2 were examined by adhesion test and flow cytometry,respectively. Results： The nucleotide and putative amino acid sequences of cloned fliR gene had 99.9% and 100% similarities to those of reported sequences in GenBank. The nucleotide sequence of the cloned kana gene was identical to the corresponding sequence in pET42a map. The results of PCR and sequencing confirmed that kana gene was inserted in the sequence of 56601^fliR-Kana fliR gene. The mRNA level of fliR gene in 56601^fliR-Kana was remarkably decreased （P 〈0.01） while the mRNA level of fliR gene in 56601^siRNA-R2 was much lower than that in wild strain 56601 （P〈0.05）. 56601^fliR-Kana and 56601^siRNA-R2 lost the ability to adhere J774A. 1 cells; and their ability to induce cell necrosis and apoptosis was markedly weakened （P 〈 0. 01 ）. Conclusion： fliR is a virulence-associated gene of L. interrogans and the function of the gene is closely related to adhesion,induction of cell necrosis and apoptosis of the microbe.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2008年第6期572-578,共7页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金资助项目(30370072)
