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目的确定假定蛋白CPSIT_0942在鹦鹉热嗜衣原体(Cps)感染细胞中的定位及特征。方法采用聚合酶链反应(PCR)方法扩增Cps cpsit_0942基因,克隆入p GEX-6p-1原核表达载体,转化至大肠杆菌E.coli BL21,IPTG诱导表达融合蛋白GST-CPSIT_0942,纯化后免疫小鼠制备抗体,间接免疫荧光(indirect immunoinfluscent assay,IFA)检测CPSIT_0942蛋白在细胞内的定位,ELISA检测其在体外诱导THP-1细胞表达炎症因子IL-1β、IL-6和TNF-α的情况。结果 CPSIT_0942蛋白能得到可溶性表达蛋白,可诱导机体产生抗体,IFA主要定位于Cps包涵体,该蛋白体外能诱导THP-1细胞产生炎症因子,在4μg/ml的浓度时,GST-CPSIT_0942诱导产生的IL-1β、IL-6和TNF-α达到最大值,且当以4μg/ml蛋白刺激细胞12 h时,IL-1β、IL-6和TNF-α达到高峰。结论 CPSIT_0942主要定位于包涵体上,能诱导THP-1细胞产生炎症因子,为阐明衣原体致病机制提供了重要的实验依据。
Objective To determine the localization and characterization of putative protein CPSIT_0942 in infected cells of C. parrot. Methods The Cps cpsit_0942 gene was amplified by polymerase chain reaction (PCR) and cloned into prokaryotic expression vector pGEX-6p-1. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The fusion protein GST-CPSIT_0942 was induced by IPTG. The antibodies were prepared by indirect immunofluorescence assay (IFA). The expression of IL-1β, IL-6 and TNF-α in THP-1 cells was detected by ELISA. Results CPSIT_0942 protein could express soluble protein and induce the body to produce antibodies. IFA was mainly located in Cps inclusion body, which could induce THP-1 cells to produce inflammatory cytokines in vitro. Under the concentration of 4μg / ml, GST-CPSIT_0942 induced IL-1β, IL-6 and TNF-α reached the maximum value, and IL-1β, IL-6 and TNF-α peaked when cells were stimulated with 4μg / ml protein for 12 hours. Conclusions CPSIT_0942 is mainly located on the inclusion body and can induce THP-1 cells to produce inflammatory cytokines. It provides an important experimental basis for elucidating the pathogenesis of Chlamydia.