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RNA interfered target sequence of HBV and use thereof

A technology of RNA interference and target sequence, which is applied in the field of molecular biology and biomedicine, can solve the problems that the inhibitory effect varies greatly, and there is no uniform determination of the most effective target.

Inactive Publication Date: 2005-09-14
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi works at the mRNA level. For a specific gene, not arbitrarily selecting a sequence can achieve the inhibitory effect. The inhibitory effect of different targets varies greatly, but so far there is no unified rule for determining the most effective target

Method used

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  • RNA interfered target sequence of HBV and use thereof
  • RNA interfered target sequence of HBV and use thereof
  • RNA interfered target sequence of HBV and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of shRNA expression vector pU6P

[0030] 129Sv / Ev mouse genomic DNA was used as a template for PCR to amplify a 276bp U6 promoter sequence. The PCR product was double-digested with EcoRI and XbaI, ligated with the pBluescript vector (Stratagene) that was also double-digested, transformed into Escherichia coli competent cells, positive clones were picked, and the plasmid was extracted for sequencing to confirm the correctness of the inserted sequence. Amplification primers are as follows:

[0031] MU6p5:

[0032] 5′-GC gaattc (EcoRI) gcggccgc (NotI)(-276)ACAGACTTGTGGGAGAAGCT-3'

[0033] MU6p3:

[0034] 5′-GGC tctaga (XbaI) g(+1)atatc (EcoRV)AAACAAGGCTTTTCTCCAAGG-3′

Embodiment 2

[0035] Example 2: Construction of pU6-siHBV vector

[0036] The genome sequences of different genotypes of HBV (23 in total) were searched from GenBank, compared with CLUSTERX software, and the conserved segments were found. According to the principle of siRNA target selection, based on the HBV sequence in 2.2.15 cells (GenBank accession number U95551), select 21-24 nt sequences in the conserved region as candidate targets, and then compare these sequences with the genome database , confirming that there is no homology with the human genome, a total of 12 target sequences were selected (Table 1, Figure 1A ). After the target sequence is determined, two complementary oligonucleotide chains are synthesized according to the requirements of vector construction. Each strand includes the sense sequence of the target sequence, the loop sequence (TTCAAGAGA), the antisense sequence of the target sequence, the transcription termination signal (TTTTT) and the sticky end sequence after ...

Embodiment 3

[0040] Example 3: Transient inhibition of HBV in 2.2.15 cells by shRNA expression plasmid

[0041] 2.2.15 Cell culture in 5% CO 2 In a 37° C. incubator, RPMI 1640 culture medium contained 10% fetal bovine serum (FBS), ampicillin 100 u / ml, streptomycin 100 μg / ml and 200 μg / ml G418. Select the transfection parameters according to the results of the optimization experiment, the main transfection steps are as follows:

[0042] On the day before transfection, digest and count the cells, dilute the cells with culture medium containing serum and no antibiotics, spread the 96-well plate, add 100 μl cell suspension to each well, so that the number of cells is about 1×10 4 For each cell, the confluency of the cells reached 40-50% during transfection; for each well of cells, dilute 240ng of plasmid DNA with 25μl of serum-free opti-MEM; and dilute 1μl of LF2000, and place it at room temperature for 5min; Mix LF2000 and plasmid DNA together, mix gently, and place at room temperature for ...

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Abstract

This invention belongs to molecule biology and biomedicine technique field. It relates to 9 target points of RNA disturbing to HBV and applications of preparing hepatitis B medicine according to these target sequence. Plasmid is expressed through shRNA and siRNA transfection cell and animal cell is chemical synthesized. Then 16 RNA target point on HBV tissue is sieved, 9 of them can obvious restrain HBV protein expression at cell level. Expression and virus duplication of HBV protein inside cell can be stably restrained by 11 target point, and expression of HBV transgene little mouse in vivo virus protein can be restrained. Medicine curing hepatitis B can be made according to these target sequence.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine, and relates to nine targets on the HBV sequence for RNA interference, and the use of siRNA obtained in various ways for these targets to prepare new drugs for treating hepatitis B . Background technique [0002] Hepatitis B virus (hepatitis B, HB) is caused by hepatitis B virus (hepatitis B virus, HBV). HBV infection can not only lead to acute and chronic viral hepatitis and severe hepatitis, but also closely related to the occurrence and development of cirrhosis (livercirrhosis, LC) and hepatocellular carcinoma (hepatocellular carcinoma, HCC). 20% of patients with chronic hepatitis B will develop liver cirrhosis, and the risk of HCC in people with chronic HBV infection is 100 times that of normal people. About 350 million people in the world are chronic carriers of HBsAg, 3 / 4 of which are in Asia. HBV infection causes 500,000 to 1,200,000 deaths worldwide each year, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P1/16C12N15/11
Inventor 徐安龙任向荣周玲君罗广彬谢珍慧林斌董美玲
Owner SUN YAT SEN UNIV
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