靶向敲除问号钩端螺旋体鞭毛相关fliN基因及其突变株致病性变化  

Targeting knock out of Leptospira interrogans flagellum-associated fliN gene and pathogenic function alteration of the mutant

作　　者：楼宏强[1,2] 廖苏梅[1] 胡野[2] 严杰[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310058 [2]浙江省金华职业技术学院医学院,321000

出　　处：《中华微生物学和免疫学杂志》2009年第8期677-682,共6页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：国家自然科学基金资助项目（30370072）

摘　　要：目的了解问号钩端螺旋体（简称钩体）鞭毛相关Ⅲ型分泌系统∥洲基因产物的致病性。方法构建基因打靶载体p2NIL fliN-amp，采用基于自杀质粒与染色体同源序列重组的靶基因敲除技术构建不表达FliN的问号钩体赖型56601株突变株，采用PCR、测序和Westernblot对FliN-突变株进行鉴定。采用动力试验、MTT、Fontana镀银染色法和流式细胞术，对FliN-突变株的迁移能力、培养物上清对小鼠单核-巨噬样细胞株J774A．1的细胞毒性、黏附及诱导J774A．1细胞凋亡能力的改变进行分析。结果PCR、测序和Westernblot结果均证实成功构建了FliN‘突变株，该突变株能在含100μg/ml氨苄青霉素的Korthof培养基中生长及繁殖。FliN。突变株在Korthof半固体培养基上菌落直径（2—3mm）明显小于野生株（6～8mm），表明该突变株动力消失。FliN一突变株培养物上清作用J774A．1细胞72h时虽显示有细胞毒性，但明显低于野生株（P〈0．05）。FliN。突变株作用J774A．1细胞60rain时的黏附率（25．5％）明显低于野生株（47．5％）（P〈0．05），其引起J774A．1细胞坏死和细胞凋亡率（30．6％和22．7％）也明显低于野生株（48．2％和35．2％）（P〈0．05）。结论．fliN基因产物与问号钩体黏附细胞、分泌毒力因子和诱导细胞凋亡密切相关。采用基于自杀质粒基因敲除技术可用于研究问号钩体靶基因产物的致病机制。Objective To determine the pathogenicity of product expressed by Leptospira interro- gans flagellum-associated tiN gene belonging to type Ⅲsecretion system. Methods Target gene knock out technique based on homologious sequence recombinant of suicide plasmid and chromosome was applied to construct FliN unexpressed mutant from L. interrogans serovar Lai strain 56601. PCR, sequencing and Western blot were used to identify FliN＂ mutant. Motility test, MTT, Fontana silver staining method and flow cytometry were respectively performed to determine the alterations of migration ability, cytotoxicity of the cul- ture supernatant to murine mononuclear-macrophage like cell line （ J77dA. 1 ）, adhering to and inducing ap- optosis of J774A. 1 cells of FliN＂ mutant. Results All the results of PCR, sequencing and Western blot confirmed a successful construction of FliN- mutant, and this mutant could grow as well as propagate in 100 μg/ml ampicillin contained Korthof medium. The diameters （2-3 mm） of FliN- mutant＇s colonies on semi- solid Korthof medium were remarkbly less than those （6-8 mm） of wild strain, indicating mobility absence of the mutant. The supernatant of FliN mutant culture treating J774A. 1 cells for 72 h showed cytotoxicity, but the cytotoxicity was significantly lower than that of wild strain （P 〈0.05）. The adhering ratio （25.5%） of FliN- mutant co-incubating with J774A. 1 cells for 60 rain was also obviously lower than that of wild strain. Furthermore, both the necrotic and apoptotic rates （30.6% and 22.7% ） induced by FliN- mutant were low- er than those （48.2% and 35.2% ） caused by wild strain （ P 〈 0.05 ）. Conclusion The product of tiN gene displays a close relationship with adhering cells, secreting virulent factors and inducing cell apoptosis of L. interrogans. The gene knock out technique based on suicide plasmid can be used to study the pathogenic mechanism of target gene products of L. interrogans.

关 键 词：问号钩端螺旋体 fliN基因 基因敲除 突变株 致病性 

分 类 号：R514[医药卫生—内科学]