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Smal linterference ribonucleic acid sequence of hepatitis B virus gene and preparing method

A technology of ribonucleic acid and hepatitis B virus, applied in the field of genetic engineering, can solve problems such as technical difficulty, RNA interference, and unsuitable for long-term research projects

Inactive Publication Date: 2007-04-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (2) Current status of RNA interference
But the technology is difficult; (3) RNaseIII degrading dsRNA method: After the target mRNA of 200-1000 bases is prepared by in vitro transcription, it is then digested in vitro with RNaseIII to obtain a mixture of various siRNAs. The advantages are: It can quickly and economically study the phenotype of a gene function loss, but it is not suitable for long-term research projects, or requires a specific siRNA for research, especially gene therapy; (4) siRNA expression vector method: construct expression-specific The carrier of siRNA can be introduced into eukaryotic cells to express siRNA, which has the advantage of being able to be amplified in large quantities, but there is a potential risk of gene integration; (5) siRNA expression cassette (siRNA expression cassettes) method: directly obtain the DNA expressing siRNA by PCR method The template is transcribed into siRNA in vivo after being introduced into eukaryotic cells. The advantage is that the method is simple and fast, and can be used to screen effective siRNA sequences, but there is also a potential risk of gene integration

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 In vitro transcription and synthesis of interfering RNA inhibits the expression of HBV surface antigen fusion gene in vitro

[0030] Preparation of siRNA

[0031] (1) Design of siRNA According to the design principle of siRNA and the experimental results of direct synthesis of siRNA, the target cDNA target sequences of 2 siRNAs targeting HBV (ayw subtype) S gene were selected. The sequence is as follows:

[0032] (anti-s siRNA1)5'-AAACCTTCGGACGGAAATTGC-3'

[0033] (anti-s siRNA2)5'-AATACCGCAGAGTCTAGACTC-3'

[0034] (2) Design and synthesis of transcription templates for specific siRNAs According to the above cDNA target sequences, two 29-mer DNAs were designed and synthesized as templates, including 8 bases matching the 3' end of the T7 promoter primer (called For the guide sequence, leader sequence) and 21 designed siRNA sequences.

[0035] ① The antisense and sense strands of specific anti-s siRNA1 are as follows:

[0036] Antisense anti-s siRNA1 Oligon...

Embodiment 2

[0074] Example 2 Anti-s siRNA inhibits the expression of green fluorescent protein and HBV S gene expression in HepG2 cells co-transfected with reporter plasmid pGFP-S and siRNA

[0075] (1) Co-transfection of reporter plasmid pGFP-S and siRNA into HepG2 cells Reporter plasmid pGFP-S is a plasmid expressing reporter gene green fluorescent protein and HBsAg fusion protein. HepG2 cells according to 1 × 10 5 Inoculate a 24-well plate per well, cultivate for 24 hours to reach a fusion rate of 80%-90%, and co-transfect pGFP-S and siRNA. For specific steps, refer to the instruction manual of Lipofectamine 2000 from Invitrogen Company. After 6 hours, change to 10% FCS DMEM, and at the same time set up a negative group transfected only with pGFP-S vector, and repeat 3 wells for each group.

[0076] (2) Observation of cell GFP expression by fluorescence microscope After 48 hours of transfection, the expression of GFP in HepG2 cells was observed under an inverted fluorescence microscop...

Embodiment 3

[0079] Example 3 Anti-s siRNA inhibits the expression of HBsAg and HbeAg in siRNA transfected HepG2.2.15 cells

[0080] (1) siRNA transfection of HepG2.2.15 cells HepG2.2.15 cells according to 1 × 10 4 Inoculate a 24-well plate, cultivate for 24 hours to achieve a fusion rate of 30%-40%, and transfect 0.84 μg of siRNA each. For specific steps, refer to the instruction manual of OLIGOFECTAMINE reagent from Invitrogen Company. Set irrelevant control siGFP group (FEBS Letters 543 (2003) 51-54): sense RNA 5-GGCUACGUCCAGGAGCGCACC-3, anti-sense RNA 5-UGCGCUCCUGGACGUAGCCTT-3. Change to 10% FCS DMEM after 6 hours. Three replicate wells were set up for each group.

[0081] (2) Detection of HBsAg and HBeAg concentration changes by radioimmunoassay At 48 hours after transfection of HepG2.2.15 cells, cell culture fluid was collected, centrifuged to remove cell debris, and the supernatant was detected by radioimmunoassay. For the detection method, refer to the kit instructions. After tra...

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PUM

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Abstract

The present invention provides small interference ribonucleic acid (siRNA) sequence of hepatitis B virus (HBV) and their preparation process. SEQ ID Nos. 1-4 are two pairs of transcription siRNA DNA template sequences of HBV (ayw subtype) S gene of the present invention. Specific siRNA for HBV (ayw subtype) S gene is firstly synthesized in extracorporeal transcription process and then used to transfect cell to reach the aim of inhibiting the expression of HBV gene. The said method can obtain great amount of HBV siRNA fast economically for HBV resisting experiment research and clinical application.

Description

technical field [0001] The invention belongs to biotechnology and relates to the technical field of genetic engineering, in particular to a small interference ribonucleic acid sequence for hepatitis B virus gene and a preparation method. Background technique [0002] (1) Current status of treatment of hepatitis B virus (HBV) [0003] Chronic hepatitis B is one of the diseases with high incidence in my country, and it is the main cause of liver fibrosis and liver cancer. The current drugs for treating hepatitis B are mainly immunomodulators based on alpha interferon and nucleoside analogues based on lamivudine and adefovir, but the therapeutic effect is still unsatisfactory. The continuous replication of HBV DNA is the main cause of chronic hepatitis B. Therefore, effective inhibition of HBV DNA replication is the key to treating chronic hepatitis B and controlling persistent HBV infection. [0004] (2) Current status of RNA interference [0005] Recently, the emergence o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12N15/51A61K48/00A61P1/16
Inventor 朱海红陈智
Owner ZHEJIANG UNIV