Dual expression vector for immunostimulatory RNA and HBV target gene silence RNA, construction and application thereof

A technology of immune stimulation and target gene, applied in the field of genetic engineering, to achieve the effect of good inhibitory effect and good application prospect

Inactive Publication Date: 2011-02-23
SHANDONG UNIV
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  • Dual expression vector for immunostimulatory RNA and HBV target gene silence RNA, construction and application thereof
  • Dual expression vector for immunostimulatory RNA and HBV target gene silence RNA, construction and application thereof
  • Dual expression vector for immunostimulatory RNA and HBV target gene silence RNA, construction and application thereof

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Embodiment

[0032] 1. Construction of a double expression vector:

[0033] (1) For the sequence of the HBV x gene, design the specific silencing RNA and the corresponding DNA sequence for the HBV x gene, and add the double restriction site sequences Sal I and Xbal I at both ends of the sequence, and anneal to form a double strand Structure, the sequence is shown in Table 1;

[0034] (2) The pTZU6+1 vector is subjected to double enzyme digestion of Sal I and Xbal I, and the vector skeleton is recovered;

[0035] (3) linking the sequence prepared in step (1) with the pTZU6+1 vector prepared in step (2) to construct pTZU-shRNA;

[0036] (4) Use cationic liposome Lipofectamine2000 (purchased from invitrogen) to transfect the constructed pTZU-shRNA into HBV-infected human liver cell line HepG2.2.15 cells, and detect the expression level of HBV x gene, and screen the expression level of the gene with better silencing sequence of abilities. Then the screened pTZU-shRNA (shRNA3) was digested w...

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Abstract

The invention discloses a dual expression vector for immunostimulatory RNA and HBV target gene silence RNA. The dual expression vector is a recombinant vector consists of a DNA sequence of transcription shRNA, the DNA sequence of transcription ssRNA and pTZU6+1 vector; and the sequence of the dual expression vector is shown as seq.1. By transfecting HepG2.2.15 cells by using the dual expression vector for the immunostimulatory RNA and the HBV target gene silence RNA of the invention, the vector can be expressed in cells by Northern blot identification; by transfecting the HepG2.2.15 cells, the vector can regulate the expression of HBV down by detecting after 24 hours; and by transfecting the HepG2.2.15 cells, the vector can regulate the HBV antigenic content and the intracellular antigen content in supernatant liquid down by detecting after 24 hours. Thus, the dual expression vector for the immunostimulatory RNA and the HBV target gene silence RNA of the invention can be used as an anti-HBV medicament.

Description

technical field [0001] The invention relates to a dual expression carrier of immune stimulating RNA and HBV target gene silencing RNA, and its construction and application, belonging to the technical field of genetic engineering. Background technique [0002] HBV infection can cause acute and chronic hepatitis, and is closely related to the pathogenesis of liver cirrhosis and liver cancer. Moreover, as a virus that can escape immune surveillance, it cannot cause an immune response in the early stage of infection, so it cannot be cleared by the body's antiviral system. The treatment of HBV has brought challenges. The use of RNAi technology in anti-HBV therapy is expected to become a revolutionary new method for the treatment of hepatitis B. Since HBV replication has the characteristics of synthesizing DNA with RNA as a template, as long as RNA synthesis is blocked, DNA replication can be inhibited, so HBV infection is particularly suitable for RNAi therapy. And certain RNA...

Claims

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Application Information

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IPC IPC(8): C12N15/117C12N15/113A61P1/16C12N15/63C12N15/66A61K48/00A61P31/20
Inventor 张彩兰培祥田志刚张建
Owner SHANDONG UNIV
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