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Defensin mNP-1 and applications thereof in preparing anti-influenza virus drugs

A technology of mnp-1 and medicine, which is applied in the direction of antiviral agents, applications, pharmaceutical formulations, etc., can solve the problem of less antiviral

Active Publication Date: 2011-10-19
北京中加保罗生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Herpes simplex virus type 2 (HSV-2), vesicular stomatitis virus (vesicular stomatitis virus), and influenza virus A / WSN (influenza virus A / WSN) are also easily infected by MCP-1 and MCP-2. Neutralizes, but MCP-1 and MCP-2 are ineffective against cytomegalovirus, echovirus type 11, and reovirus type 3
[0038] The antibacterial activity of NP-1 has been reported more (Patterson-Delafield et al., 1980, 1981; Selsted et al., 1984, 1985c; Lehrer et al, 1983, 1985a, 1985b, 1986; Levitz et al., 1986 ; Miyasaki et al., 1990b, Borenstein et al., 1991a, 1991b; Kohashi et al., 1992), but there are fewer reports on antiviral aspects (Nakashima et al., 1993; Lehrer et al, 1985b; Sinha et al., 2003), there is no report on the anti-avian influenza virus

Method used

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  • Defensin mNP-1 and applications thereof in preparing anti-influenza virus drugs
  • Defensin mNP-1 and applications thereof in preparing anti-influenza virus drugs
  • Defensin mNP-1 and applications thereof in preparing anti-influenza virus drugs

Examples

Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1. Expression vector construction method of Chlorella ellipsoides nitrate reductase mutant

[0068] Primers were designed according to the Ubiquitin promoter sequence (SEQ ID NO: 1), upstream primer: 5' CCGGAAGCTT GTGCAGCGTGACCCG3' (SEQ ID NO: 2); downstream primer: 5' GCCCGGATCC CTGCAGAAGT3' (SEQ ID NO: 3), wherein a Hind III restriction site (underlined part) is added to the upstream primer, and a BamH I restriction site (underlined part) is added to the downstream primer, and PCR method is used to obtain The Ubiquitin promoter was obtained in the maize genome, and the sequencing results showed that it was a Ubiquitin promoter sequence (SEQ ID NO: 4, which added Hind III restriction sites and BamH I respectively at the 5' end and 3' end of SEQ ID NO: 1 Restriction sites). The PCR reaction conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and extension at 72°C for ...

Embodiment 2

[0069] Example 2. Using the pGreen0029 framework to construct an expression vector.

[0070] Design primers according to Nos terminator sequence (SEQ ID NO: 6), upstream primer: 5' ATAAGAATGCGGCCGC TCGAATTTCCCCGATCGTTCAAAC 3' (SEQ ID NO: 7) downstream primer: 5' CGAGCTCG CCCGATCTAGTAACATAGATGA3' (SEQ ID NO: 8) wherein a Not I restriction site (underlined part) is added to the upstream primer, a Sac I restriction site (underlined part) is added to the downstream primer, and the PCR method is used The Nos terminator was obtained from pBI221 (Clontech). The PCR reaction conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and extension at 72°C for 10 min after 30 cycles. The 268bp fragment of the PCR product was double-digested with Not I and Sac I endonucleases, and the plasmid pGreen0029( image 3 , from BBSRC) were also double-digested with Not I and Sac I endonucleases, and then the two...

Embodiment 3

[0071] Example 3. Construction of the expression vector pGreen-NR-U-mNP1 of Chlorella ellipsoides nitrate reductase mutant.

[0072] According to conventional techniques, the Ubiquitin promoter (SEQ ID NO: 1) was extracted from the vector pbinUGUS ( figure 2) after being recovered by HindIII and BamHI double enzyme digestion, connected to the pGreen0029nos vector that was also subjected to HindIII and BamHI double enzyme digestion ( image 3 , from BBSRC) to obtain pGreen0029-U-nos primary intermediate vector ( Figure 5 ). The sequence with the target gene mNP-1 (its sequence is as SEQ ID NO: 13) was synthesized by chemical synthesis method. In order to improve the antiviral activity of NP-1, in the process of synthesis, the N-terminal of NP-1 added A methionine transforms NP-1 into mNP-1. Because the mNP-1 gene is too small, in order to facilitate the construction of the vector, a 75bp auxiliary sequence was added to the C-terminus of the gene. Two restriction sites BamH...

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Abstract

The invention relates to defensin mNP-1 which utilizes chlorella to express modified rabbit defensin NP-1(Mnp-1), the defensin expressed in the chlorella has strong inhibition or skilling effect on bird flu virus H5N1 and H9N2. The result of the invention can be applied for preparing anti-influenza virus drugs.

Description

technical field [0001] The present invention relates to defensin and its application in preparation of anti-influenza virus medicine. Specifically, it relates to the defensin mNP-1 produced by Chlorella, and its application in the preparation of anti-influenza virus drugs. Background technique [0002] Structural Features and Classification of Defensins [0003] Defensins are a class of cationic small peptides widely present in animals, plants and humans with microbial resistance. Defensins generally consist of 29-54 amino acids with a molecular weight of 3-6KD. It has the following common characteristics in structure: (1) positively charged, (2) rich in arginine, (3) has a certain number of conserved cysteine, (4) forms intramolecular molecules through cysteine ​​molecules Disulfide bonds, cyclization of peptides to form antiparallel β-sheet structures. Since Professor Lehrer of the University of California named it in 1985 (Selsted et al., 1985), it has received extens...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/79C12N1/13A61K38/17A61K48/00A61P31/12A61P31/14A61P31/16
Inventor 胡赞民陈宇红尹维波白丽莉孙勇如宋丽英赵世民陈凡储成才张克臣曹国治吴东来关云涛
Owner 北京中加保罗生物科技有限公司
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