沙眼衣原体pORF5稳定转染HeLa细胞后宿主蛋白转录谱分析

Analysis of differential protein expression profile of HeLa cells stably transfected with Chlamydiatrachomatis pORF5 gene

目的分析沙眼衣原体（Chlamydia trachomatis，Ct）质粒蛋白pORF5基因对HeLa细胞蛋白质表达谱的影响，为深入研究Ct致病机制提供实验依据。方法构建pORF5基因慢病毒表达载体，与辅助质粒共转染293T细胞包装慢病毒颗粒，收集慢病毒后感染HeLa细胞，流式细胞术多次分选获得pORF5基因稳定转染的HeLa细胞系（pORF5-HeLa细胞系）和对照HeLa细胞系。采用同位素标记相对和绝对定量（iTRAQ）技术结合二维液相色谱串联质谱（2DLC-MS/MS）技术建立pORF5-HeLa细胞和对照HeLa细胞蛋白质表达谱，筛选并构建差异蛋白质表达数据库，采用qRT-PCR和Western blot方法对部分差异蛋白质进行验证。结果成功建立了稳定过表达pORF5基因的HeLa细胞系和对照细胞系；采用iTRAQ结合2D LC-MS/MS质谱技术共鉴定了314个差异表达的宿主蛋白质，其中有159个蛋白质表达上调，155个蛋白质表达下调。差异蛋白质主要涉及代谢过程、免疫应答、生物黏附等众多事件。pORF5基因转染的HeLa细胞中组蛋白H1.2C（HIST1H1C）、血红蛋白α亚基（HBA1）、帕金森蛋白（PARK7）、高迁移率族蛋白B1（HMGB1）、HMGB2的mRNA表达水平上调，而胞内氯离通道蛋白1（CLIC1）、细胞角蛋白7（KRT7）、SFN（14-3-3σ）、周期素依赖性刺激抑制因子2A（CDKN2A）的mRNA表达水平下调，Western blot证实PARK7、HMGB1蛋白表达水平增加，这些结果均与蛋白质组学结果一致。结论成功构建了pORF5基因稳定转染HeLa细胞的差异蛋白质表达谱，发现了一组受pORF5基因调控并与细胞代谢、增殖和黏附等生物学过程相关的差异表达蛋白质。提示pORF5可能通过改变宿主蛋白质的表达，影响宿主细胞生物学行为以促进Ct的生长发育。

ObjectiveTo analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis.MethodsA lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation （iTRAQ） approach combined with nano-liquid chromatography-tandem mass spectrometry （NanoLC-MS/MS） analysis was performed to understand protein expression profiles and to identify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells （pORF5-Hela） and the control HeLa cells. Quantitative real-time PCR （qRT-PCR） and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively.ResultsHeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successfully. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C（histone H1.2C）, HBA1（hemoglobin subunit alpha）, PARK7（parkinson disease protein 7）, HMGB1（high mobility group protein B1） and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1（chloride intracellular channel protein 1）, KRT7（typeⅡ cytoskeletal 7）, SFN（14-3-3 protein sigma） and CDKN2A（cyclin-dependent kinase inhibitor 2A） were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at protein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data.ConclusionExpression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.