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Inhibition of Hepatitis Bvirus Expression and Replication by ASGPR-directed Delivery of SiRNA

Author: MaZuoZuo
Tutor: ShenGuanXin
School: Huazhong University of Science and Technology
Course: Immunology
Keywords: Hepatitis B virus RNA interference HepG2.2.15 cells Asialoglycoprotein receptor Gene targeting
CLC: R346
Type: Master's thesis
Year: 2009
Downloads: 16
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Abstract

[Objective] To design and build a different sequence-specific siRNA vector against hepatitis B virus (HBV) gene to explore the role of siRNA HepG2.2.15 cells HBV replication and expression. Screened efficient silencing of HBV carrier, to go to the sialic acid receptor targeting targeted HepG2.2.15 cells observation the targeted silencing effect, as well as cell activity, to lay the foundation for further in vivo targeting studies. 【Methods】 1 siRNA eukaryotic vector construction: design and synthesis of the six pairs of different sequence of the HBV genome siRNA oligonucleotides were annealed to form double-stranded, and then treated with T4 ligase, cloned into the expression of EGFP true The nuclear carrier, named pGenesil-siHBV1 ~ 6, and the establishment has nothing to do with the HBV sequence pGenesil-siHBV-HK as a negative control. 2. Biological activity of the recombinant identification: The recombinant plasmid pGenesil-siHBV1 ~ 6 and pGenesilsiHBV-HK liposomes were transfected HepG2.2.15 cells. Using real-time quantitative PCR 2d, 3d, 4d target gene mRNA after transfection and cell culture supernatant cccDNA level; the ELISA detection transfer dye 2d, 3d, 5d, 7d cell culture supernatant HBsAg, HBeAg expression . Desialylated receptors targeting: efficient silencing of HBV siRNA vector using jetPEI-Hepatocyte targeting HepG2.2.15 cells transfected with inverted difference EGFP fluorescence microscopy and flow cytometry (FCM) to detect cell expression to detect the transfection efficiency; FCM to detect cell the HBcAg expression as well as after transfection the 3d cell apoptosis; The ELISA detection cell culture supernatant HBsAg, HBeAg expression, cell immunochemical ELISA technique to observe intracellular expression of HBsAg. [Results] The Construction of HBV-specific siRNA vector: PCR, restriction analysis and sequencing results showed that HBV gene-specific siRNA eukaryotic expression vector was successfully constructed. Biological activity of HBV-specific siRNA carrier: Realtime-PCR, ELISA, respectively from the mRNA, protein, and the cccDNA level of detection, the results show that the pGenesil-siHBV1 ~ 6 can be varying degrees of inhibition of HBV replication and antigen expression, which pGenesil- siHBV1 the strongest inhibitory effect. 3. ASGPR targeting pGenesil-siHBV1 for HBV silencing effect: pGenesil-siHBV1 to jetPEI-Hepatocyte targeting transfected HepG2.2.15 cells, the inverted difference visible green fluorescence under a fluorescence microscope, without jetPEI-Hepatocyte transfection control group did not See the green fluorescent FCM detection of green fluorescence positive cells was approximately 45%; the HBcAg expression of FCM to detect cell and ELISA detection of the targeted experimental group cell culture supernatant HBsAg, HBeAg expression the lower than LipofectamineTM2000 transfected control group, and the two the control group were significantly lower than the unrelated sequence; cell immunochemical ELISA technique to observe intracellular expression of HBsAg unrelated sequence of the control group significantly reduced; FCM detection turn after transfection the 3d of apoptosis was no significant change. [Conclusion] The results show that there are differences for HBV different siRNA silencing effect; ASGPR mediated targeted delivery of siRNA vectors can better play the role of silent HBV replication and expression of the gene, and laid a good foundation for the next step in the in vivo experiments .

Inhibition of Hepatitis Bvirus Expression and Replication by ASGPR-directed Delivery of SiRNA

Abstract

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