CAJ | 학술논문

目的：研究鹦鹉热嗜衣原体（ Chlamydophila psittaci，Cps） CPSIT＿0271蛋白的定位和表达时相，及其重组蛋白对人单核细胞（ THP-1）产生促炎症因子的诱生作用。方法原核表达、纯化GST-CPSIT＿0271重组蛋白，免疫BALB/c小鼠制备多克隆抗体，ELISA分析血清抗体滴度。间接免疫荧光法初步观察内源性CPSIT＿0271的定位，并通过Western blot 分析CPSIT＿0271在Cps感染HeLa细胞8 h、18 h、24 h、36 h和48 h后的表达情况。用不同浓度GST-CPSIT＿0271刺激THP-1细胞，ELISA检测IL-6、IL-1β和TNF-α水平。结果在Cps感染的HeLa细胞中，CPSIT＿0271分布于衣原体包涵体内。 CPSIT＿0271在Cps感染HeLa细胞后36 h开始表达，在48 h时表达量增加。 GST-CPSIT＿0271免疫小鼠，产生较强的免疫应答，小鼠血清特异性抗体效价为1∶16000。当GST-CPSIT＿0271蛋白浓度为2～5μg/ml时，刺激THP-1细胞产生IL-6、IL-1β和TNF-α的水平与CPSIT＿0271呈明显的剂量依赖关系。5μg/ml的GST-CPSIT＿0271蛋白刺激THP-1细胞，TNF-α和IL-1β在24 h时达到高峰，IL-6在48 h时达到高峰。结论 CPSIT＿0271蛋白分布于包涵体内，CPSIT＿0271基因可能为Cps晚期表达基因；GST-CPSIT＿0271具有较好的免疫原性，并能促进THP-1细胞分泌促炎症因子IL-6、TNF-α和IL-1β。
목적：연구앵무열기의원체（ Chlamydophila psittaci，Cps） CPSIT＿0271단백적정위화표체시상，급기중조단백대인단핵세포（ THP-1）산생촉염증인자적유생작용。방법원핵표체、순화GST-CPSIT＿0271중조단백，면역BALB/c소서제비다극륭항체，ELISA분석혈청항체적도。간접면역형광법초보관찰내원성CPSIT＿0271적정위，병통과Western blot 분석CPSIT＿0271재Cps감염HeLa세포8 h、18 h、24 h、36 h화48 h후적표체정황。용불동농도GST-CPSIT＿0271자격THP-1세포，ELISA검측IL-6、IL-1β화TNF-α수평。결과재Cps감염적HeLa세포중，CPSIT＿0271분포우의원체포함체내。 CPSIT＿0271재Cps감염HeLa세포후36 h개시표체，재48 h시표체량증가。 GST-CPSIT＿0271면역소서，산생교강적면역응답，소서혈청특이성항체효개위1∶16000。당GST-CPSIT＿0271단백농도위2～5μg/ml시，자격THP-1세포산생IL-6、IL-1β화TNF-α적수평여CPSIT＿0271정명현적제량의뢰관계。5μg/ml적GST-CPSIT＿0271단백자격THP-1세포，TNF-α화IL-1β재24 h시체도고봉，IL-6재48 h시체도고봉。결론 CPSIT＿0271단백분포우포함체내，CPSIT＿0271기인가능위Cps만기표체기인；GST-CPSIT＿0271구유교호적면역원성，병능촉진THP-1세포분비촉염증인자IL-6、TNF-α화IL-1β。
Objective To study the intracellular localization and temporal expression of CPSIT_0271 in Chlamydia psittaci-infected cells; and to investigate the effects of recombinant GST-CPSIT_0271 protein on the expression of proinflammatory cytokines including IL-6, IL-1βand TNF-αby THP-1 cells.Methods The gene encoding CPSIT_0271 of Chlamydia psittaci was expressed as fusion protein ( GST-CPSIT_0271 ) in E.coli.The polyclonal antibody was prepared by immunizing BALB /c mice with the purified recombinant pro-tein.Antibody titer was determined by ELISA .Indirect immunofluorescence assay ( IFA) was performed to lo-cate the endogenous CPSIT_0271 protein in C.psittaci-infected cells .The expression characteristics of CPSIT_0271 protein were detected by Western blot in C.psittaci-infected HeLa cells at different time points .The levels of IL-6, IL-1βand TNF-αwere analyzed by ELISA after stimulating THP-1 cells with different concentrations of CPSIT_0271 protein.Results CPSIT_0271 protein was found to express in the chlamydia inclusion of C.psittaci-infected HeLa cells .The expression of CPSIT_0271 protein was detected firstly at 36 h and increased at 48 h after C.psittaci infection.The titer of anti-CPSIT_0271 specific antibody in GST-CPSIT_0271 immu-nized mice reached to 1 ∶16 000.GST-CPSIT_0271 protein increased the levels of IL-6, IL-1βand TNF-αin THP-1 cells in a dose-dependent manner in the range of 2 to 5 μg/ml.The levels of TNF-αand IL-1βreached their peaks at 24 h, and IL-6 level peaked at 48 h upon the stimulation by 5 μg/ml of GST-CPSIT_0271 pro-tein.Conclusion CPSIT_0271 expressed in inclusion bodies of Chlamydia psittaci in the infected cells , sug-gesting it might be a late expression gene .GST-CPSIT_0271 protein shows good immunogenicity and enhances the expressions of IL-6, IL-1βand TNF-αin THP-1 cells.