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External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation

A technology of external guidance and nucleotide sequence, which is applied in the application field of preparing anti-HCMV drugs, can solve problems such as high cost, research and application obstacles, and achieve the effect of high drug resistance and cost reduction

Inactive Publication Date: 2009-06-24
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The length of these EGS is about 54 bases, and the base length of these EGS molecules is too long. Although it can effectively and specifically silence the expression of the UL49 gene, it requires a high cost when it is used as a drug for research and development and application. The application brings great obstacles, so it is necessary to seek a shorter EGS molecule for the development of anti-HCMV drugs

Method used

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  • External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation
  • External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation
  • External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The acquisition of embodiment 1 EGS

[0026] 1. Search for candidate EGS molecules in the mRNA sequence of the HCMV UL49 gene, and the search principles are as follows:

[0027] a. Search for UUCA or UUCG sequences in the target gene (UL49 gene) mRNA sequence;

[0028] b. The base anchoring the cleavage site within 20 to 25 bases upstream of the UUCR sequence is G;

[0029] c. The loop of the candidate EGS molecule must consist of 8-9 nucleotides;

[0030] d. The Steml of the candidate EGS molecule contains at least 1 G / C base pair, preferably between 3 and 4;

[0031] e. The candidate EGS sequence contains at least 7 to 8 base pairs;

[0032] f. The number of nucleotides between Stem1 and stem2 of the candidate EGS molecule and the target gene is preferably 5-6;

[0033] g. The Tm value of the candidate EGS molecule is around 50 degrees;

[0034] h. The binding position between the candidate EGS molecule and the target gene has less secondary structure.

[0035] ...

Embodiment 2

[0036] Example 2 Verification of the establishment of the EGS gene silencing efficiency platform

[0037] In this example, the UL49 gene was first cloned using a vector, and then a cell line capable of stably expressing UL49 was constructed. The vectors, cell lines, enzymes, and various reagents used in the example were all commercially available products. For example, pcDNA3.1(+ ) / myc plasmid, the cell line can choose Hela cell line, double enzyme digestion can use BamHI and XhoI, etc.

[0038] 1. Acquisition of UL49 gene

[0039] The UL49 gene can be amplified by PCR from the genome of the known sequence, for example, the UL49 gene can be amplified by PCR from the genome of the AD169 virus strain.

[0040] The total volume of the PCR reaction system is 100 μl: LA Taq enzyme 1 μl, reaction buffer 50 μl, AD169 genomic DNA template 5 μl, UL49 upstream primer (nucleotide sequence as shown in SEQ ID NO: 3) 5 μl, UL49 downstream primer (nucleotide sequence as shown in SEQ ID NO:...

Embodiment 3

[0053] Example 3 EGS silences UL49 gene expression

[0054] The experimental group in this example is: the EGS prepared in Example 1 was transfected into the Hela cell line stably expressing the UL49 gene constructed in Example 2.

[0055] The control group of this embodiment is: (1) mutation control group: the mutant EGS prepared in Example 1 was transfected into the Hela cell line stably expressing the UL49 gene constructed in Example 2 according to the same transfection conditions as the experimental group; (2) Nonsense control group: the nonsense EGS prepared in Example 1 was transfected into the Hela cell line stably expressing the UL49 gene constructed in Example 2 according to the same transfection conditions as the experimental group; (3) Positive control group : the Hela cell line stably expressing UL49 constructed in Example 2, but not transfected with any EGS; (4) transfection reagent control group: refers to the stably expressing UL49 gene constructed in Example 2 ...

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Abstract

The invention discloses an EGS of 12nt, which can conduct RNase P ribozyme and comprises EGS which is based on RNA and has the nucleotide sequence to be shown in SEQ ID NO: 1, and EGS that is based on DNA and has the nucleotide sequence to be shown in SEQ ID NO: 2. The EGS can effectively inhibit the expression of HCMV UL49 gene. The EGS with the characteristic of DNA has the silent gene efficiency of 99%, and the EGS with the characteristic of RNA has the silent gene efficiency of 98%. Compared with the existing EGS which can cause the UL49 gene to be silent, the EGS of the invention has the sequence which only contains 12 basic groups, thus not only having the shortest nucleic acid molecules, but also effectively and specially leading the expression of the UL49 gene to be silent. The EGS of the invention is used for researching and developing the anti-HCMV drug, and can greatly reduce the cost when the drug is researched and applied.

Description

technical field [0001] The invention belongs to the field of molecular biology and medicine, and specifically relates to an external guide sequence for guiding RNase P ribozyme and its application in preparing anti-HCMV medicines. Background technique [0002] RNase P is a nucleoprotein complex widely present in various cells, which can specifically cleave the 5' end of tRNA precursor (ptRNA) in vivo to form mature tRNA. [0003] RNase P has the characteristics of recognizing the secondary structure of the substrate rather than the nucleotide sequence of the substrate. Its model substrate must include at least two elements: 1. Free CCA-3' end; 2. Specific complementary double chain area. Therefore, as long as two RNA molecules form a complex similar to the molecular structure of ptRNA, they can be recognized and cut by RNase P. [0004] The RNA molecule capable of guiding RNase P to recognize the cleavage site on the target mRNA is called an external guide sequence (Extern...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K31/7088A61P31/22C12N15/113
Inventor 周天鸿曾志锋李弘剑李月琴张欣
Owner JINAN UNIVERSITY
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