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Small interfering RNA for restraining SARS corona virus M protein gene expression, encoding gene and application thereof

A coronavirus, gene expression technology, applied in DNA/RNA fragments, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problem that siRNA is rarely reported

Inactive Publication Date: 2008-05-07
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been many reports about siRNA that can induce the interference effect against SARS virus gene RNA, thereby inhibiting the replication and protein expression of the virus and forming a protective effect on cells. However, there are few reports on siRNAs that use vectors to function

Method used

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  • Small interfering RNA for restraining SARS corona virus M protein gene expression, encoding gene and application thereof
  • Small interfering RNA for restraining SARS corona virus M protein gene expression, encoding gene and application thereof
  • Small interfering RNA for restraining SARS corona virus M protein gene expression, encoding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1, the design of the small interfering RNA that suppresses SARS-CoV M protein gene expression and the construction of RNAi interference vector

[0030] 1. Design of small interfering RNA that inhibits SARS-CoV M protein gene expression

[0031] Design the small interfering RNA that suppresses SARS-CoV M protein gene expression as follows:

[0032]According to the specific requirements of the SARS-CoV M gene mRNA sequence (GenBank number: 52100973) and the U6 promoter in the pBS / U6 vector (SuiGC et al.PNAS.2002 April; 99(8):5515-5520), the SARS - Two sequences of 22 bp and 21 bp in length at 220 and 460 bp downstream of the transcription start site aug of the CoV M gene mRNA sequence (see Figure 1 for a schematic diagram of the position of the targeting sequence in the SARS-CoV M gene sequence), avoid The mutation points between different virus strains were identified. At the same time, the two selected sequences both started with GGG, and the GC content was ...

Embodiment 2

[0054] Example 2, Detecting the dose effect of siRNA-M1 and siRNA-M2 on the inhibition of SARS-CoV M protein gene expression

[0055] 1. Construction of the expression vector pCMV-Myc-M containing the SARS-CoV M protein gene

[0056] Using the SARS-CoV M protein gene as a template, the cds region (coding sequence) of the SARS-CoV M protein gene was amplified by PCR under the guidance of primers P1: 5'-tatagaattctggcaacggtactatt-3' and P2: 5'-tataggtaccgtcacttactgtactagcaaagc-3' , and respectively added restriction endonuclease Hind III and BamH I recognition sites at both ends of the sequence, after the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and the result was a DNA with a size of about 670bp Fragment, reclaim and purify this object fragment, then use restriction endonuclease Hind III and BamHI this object fragment to carry out double enzyme digestion to the eukaryotic expression vector pCMV-Myc (purchased from BD Biosciences co...

Embodiment 3

[0066] Embodiment 3, detecting the specificity of siRNA-M1 and siRNA-M2 to SARS-CoV M protein gene expression inhibition

[0067] 1. Construction of the expression vector pCMV-Myc-M containing the SARS-CoV M protein gene

[0068] The cds region of the SARS-CoV M protein gene is amplified by PCR using the same method as in Example 2, and restriction endonuclease HindIII and BamH I recognition sites are respectively added at the two ends of the sequence, and the PCR amplification product is analyzed after the reaction. Carry out 1% agarose gel electrophoresis detection, the result has obtained the DNA fragment of size about 670bp, reclaim and purify this target fragment, then carry out double enzyme digestion with restriction endonuclease Hind III and BamH I this target fragment with The eukaryotic expression vector pEGFP-N1 (purchased from BD Biosciences) containing the green fluorescent protein (green fluorescent protein, GFP) gene was ligated by the same enzyme to obtain the ...

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Abstract

The invention discloses a small interfering RNA limiting SARS coronavirus M protein gene expression and the coding gene and the application. The small interfering RNA limiting SARS coronavirus M protein gene expression is about at least one double stranded RNA sequence among the following 1) and 2): 1). Nucleotide sequence for sequence 1 in sequence list is arranged for the sense strand; nucleotide sequence for sequence 2 in sequence list is arranged for the antisense strand; 2). Nucleotide sequence for sequence 3 in sequence list is arranged for the sense strand; nucleotide sequence for sequence 4 in sequence list is arranged for the antisense strand. The invention has an advantage of playing an important role in preparing drugs using small interfering RNA limiting SARS coronavirus M protein gene expression or an expression vector carrying the coding gene for small interfering RNA limiting SARS coronavirus M protein gene expression as active component.

Description

[0001] technical field [0002] The present invention relates to small interfering RNA and its coding gene and application, in particular to the small interfering RNA and its coding gene for inhibiting the expression of SARS coronavirus M protein gene and its application in the preparation of SARS therapeutic medicine. Background technique [0003] Severe Acute Respiratory Syndrome (SARS) is an infectious disease that seriously threatens human health and even life. SARS-CoV, the pathogen that causes SARS, is a new type of coronavirus, which has several main proteins such as spike protein S (spike), matrix protein M (matrix), envelope protein E (Envelope) and nucleoprotein N (Nuclear protein). Structural protein (Holmes KV. SARS-associated coronavirus. N Engl J Med, 2003, 348(20): 1948-51.). Among them, M protein is the main structural protein in the envelope, which may be related to virus assembly and intracellular budding, and plays an important role in virus infection. Cur...

Claims

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Application Information

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IPC IPC(8): C12N15/11C07H21/02C12N15/63C12N5/10A01K67/00A61K31/7088C12N15/113
Inventor 王颖刘力王树蕙张云
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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