Abstract: Yang Xiaoyu, Zou Yan, Gong Silu, Bu Jichang, Zhou Zhou, Liu Liangzhuan, Li Zhongyu Institute of Pathogenic Biology, Medical College, University of South China, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Hengyang, 421001, China Corresponding author: Li Zhongyu, Email: lzhy1023@hotmail.com 【Abstract】 Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid?encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor?alpha （TNF?α）. Methods The recombinant lentiviral vector containing pORF5 gene and helper plasmids were co?transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5?expressing HeLa （pORF5?HeLa） cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20 μg/L TNF?α and fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real?time PCR to measure the mRNA of Caspase3, Bax and Bcl?2, and Western blot analysis to determine the protein of Bax and Bcl?2. Results After 6?hour treatment with TNF?α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly?refractive blue apoptotic bodies in the pORF5?HeLa cells and control HeLa cells. The pORF5?HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup （pORF5?HeLa cells: 35.5% ± 4.5% vs. 9.5% ± 1.5%, t = 13.53, P < 0.01; control HeLa cells: 63.6% ± 5.8% vs. 7.9% ± 0.9%, t = 32.36, P < 0.01）. Compared with treated control HeLa cells, treated pORF5?HeLa cells showed significant decreases in mRNA of Bax （72.8%） and Caspase 3 （84.5%） （t = 35.29, 42.25,respectively, both P < 0.01）, as well as in Bax protein （t = 17.58， P < 0.01）, but significant increases in Bcl?2 mRNA and protein （6.8 times） （t = 87.12, 18.93, respectively, both P < 0.01）. Conclusion pORF5 plasmid protein can inhibit TNF?α?induced HeLa cell apoptosis likely by increasing the of anti?apoptotic protein bcl?2 and decreasing the of pro?apoptotic proteins Caspase?3 and Bax.