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The Study on The Immune Response Induced by Expressing Recombinant Plasmid of Dengue Virus Type 2 ns3 protein

Wuhan University Journal of Natural Sciences 2000年第2期5卷 245-248页

作者： LI Xiang qun 1, MAO Lin 1 , YAN Zhan qiu 1 JIANG Li feng 2, YAN Hui jun 2, GUO Hui yu 21. Deptartement of Clinical Virology, Institute of Viral Research,Hubei Medical University, Wuhan. 430071 2. Deptartement of Microbiology, Sun yat Sen Uni Deptartement of Clinical Virology Institute of Viral ResearchHubei Medical University Wuhan. 430071 Deptartement of Microbiology Sun yat-Sen University of Medical Science Guanzhou 510089

The PSV?ns3, an expressing recombinant plasmid of dengue virus type 2 ns3 protein, was injected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1∶5 120 in experimental group, but it couldn’t be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for ns3 protein. Those results Suggested that inoculation Balb/C mice with PSV?ns3 could inducing immune response, and the ns3 protein might be used as the candidate protein of DNA vaccine of dengue virus.

关键词： Key words Dengue Virus Type 2 ns3 protein DNA Vaccine Immune Response

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In vitro inhibitory analysis of consensus siRNAs against ns3 gene of hepatitis C virus 1a genotype

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Asian Pacific Journal of Tropical Medicine 2017年第7期10卷 763-770页

作者： Imran Shahid Waleed Hassan Al Malki Mohammed Wanees Al Rabia Mohammed Hasan Mukhtar Shaia Saleh R.Almalki Saad Ahmed Alkahtani Sami S.Ashgar Hani S.Faidah Muhammad Hassan Hafeez Department of Pharmacology and Toxicology College of PharmacyUmm Al Qura University Department of Medical Microbiology College of MedicineKing Abdul Aziz University Department of Biochemistry Faculty of MedicineUmm Al Qura University Department of Laboratory Medicine Faculty of Applied Medical SciencesAlbaha University College of Pharmacy Najran University Department of Microbiology College of MedicineUmm Al Qura University Department of Gastroenterology and Hepatology Fatima Memorial College of Medicine and Dentistry

Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against ns3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing ns3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The ns3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed ns3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA ns3-is44 showed maximum inhibition of ns3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA ns3-is44 exhibited 70%(Pns3-is33, which showed a 64%(Pns3-is33 + ns3-is44) decreased viral load by 84%(Pns5Bis88 + ns3-is33) targeting different region of HCV genom

关键词： Hepatitis C virus ns3 protein Stable Huh-7 cell culture system RNA interference ns5B HCV therapeutics

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