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SiRNA sequence of human cytomegalovirus UL54 gene and applications

A human cytomegalovirus and sequence technology, applied in DNA/RNA fragments, antiviral agents, recombinant DNA technology, etc., can solve problems such as interference with siRNA recognition, complex secondary structures, etc.

Inactive Publication Date: 2008-12-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

siRNA works by recognizing specific mRNA target sequences, which may have complex secondary structures, or be hidden within highly folded regions of the RNA sequence, or even form tight complexes with proteins, thereby interfering with siRNA recognition

Method used

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  • SiRNA sequence of human cytomegalovirus UL54 gene and applications
  • SiRNA sequence of human cytomegalovirus UL54 gene and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 siRNA eukaryotic expression vector inhibits the expression of UL54-EGFP fusion protein in vitro

[0014] 1. Design of siRNA: According to the design principle of siRNA, search for the AA sequence from the 100nt downstream of the UL54 mRNA start codon AUG, and the 19nt sequence adjacent to its 3' end as a candidate target, and select the one with a GC content of 40-55% The siRNA sequence was compared with the human genome sequence through the Blast function of the GenBank database to ensure no homology. The cDNA sequence of the finally selected siRNA is 5'-CTCGATGAAGTCAAGAAGT-3', the GC content is 42.1%, corresponding to the 3058-3076 nucleotide site in UL54 mRNA.

[0015] 2. Synthesis and preparation of shDNA required for expression of siRNA: the positive-sense strand sequence of shDNA required for expression of siRNA is:

[0016] 5'-CCGGT CTCGATGAAGTCAAGAAGT CTCGAG ACTTCTTGACTTCAT CGAG TTTTTG-3',

[0017] The antisense strand sequence is:

[0018] 5'...

Embodiment 2

[0029] Embodiment two siRNA expression plasmid suppresses the experiment of HCMV UL54 expression in MRC-5 cell

[0030] 1. Transfection of siRNA expression plasmid into MRC-5 cells: Human embryonic lung fibroblast MRC-5 cells were inoculated into 12-well cell culture plate, and after 24 hours of culture, the siRNA expression plasmid was used to transfect the cells with Invitrogen Lipofectamine 2000 reagent. The operation method refers to the reagent Instructions, a negative control group (transfected with an empty plasmid pGCL-GFP) and a blank control group (transfected with no plasmid) were set up at the same time.

[0031] 2. Infection of MRC-5 cells with HCMV standard strain AD169: 24 hours after plasmid transfection, MRC-5 cells were infected with HCMV standard strain AD169, and a control group not infected with the virus was established at the same time.

[0032] 3. Fluorescent quantitative RT-PCR detection of UL54 mRNA expression in MRC-5 cells: 48 hours after virus infe...

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Abstract

The invention provides a cDNA sequence of siRNA aiming at a cytomegalovirus UL54 gene of human, and the nucleotide sequence is: 5`-CTCGATGAAGTCAAGAAGT-3`. According to the design principle of the siRNA, the cDNA sequence of the selected siRNA has a GC content of 42.1 percent, and the selected siRNA is corresponding to 3058-3076 nucleotide sites in UL54 mRNA, siRNA eukaryotic expression vectors aiming at the HCMV UL54 (DNA polymerase) gene are constructed, and plasmids are extracted after transforming Escherichia coli for respectively transfecting AD293 cells and MRC-5 cells, thereby finally screening out one siRNA sequence that can effectively inhibit the UL54 gene expression, and the sequence can be applied to the preparation of drugs for curing cytomegalovirus infection of human.

Description

technical field [0001] The invention belongs to biotechnology and relates to the technical fields of molecular biology and genetic engineering, in particular to the design and screening of siRNA sequences for human cytomegalovirus (HCMV) UL54 gene and its application for inhibiting human cytomegalovirus in vivo and in vitro. Background technique [0002] Human cytomegalovirus (human cytomegalovirus, HCMV) infection is very common in the population, and the adult serum antibody positive rate can reach more than 80%. HCMV can remain latent in the body for a long time after the initial infection, and even carry the virus for life. In people with immature or deficient immune functions (such as AIDS, organ transplant patients, etc.), latent HCMV can be activated and cause serious diseases. At the same time, HCMV is also one of the most common pathogens of human congenital infection, which can cause miscarriage, stillbirth, congenital deformity, hearing impairment, developmental d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K31/713A61P31/20C12N15/113
Inventor 尚世强陶然赵正言胡妙凤段群军
Owner ZHEJIANG UNIV
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