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Alexin and application thereof to preparation of antibacterial medicament

A technology of drugs and bacteria, applied in the field of defensins, can solve the problem of high incidence

Active Publication Date: 2012-07-25
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, influenza virus A / WSN (influenza virus A / WSN) are also susceptible to MCP-1 and MCP-2 Directly neutralizes, but MCP-1 and MCP-2 are ineffective against cytomegalovirus, echovirus type 11, and reovirus type 3
In 2005, a survey by Uehnert and others in 475 hospitals in the United States showed that the inpatients with MRSA infection increased by 10 times compared with 1995, tripled in 2000, and increased by 30% compared with 2004. differences and defects, the actual incidence may be higher (Jarvis et al., 2007)
However, it has not been reported in the aspect of anti-drug-resistant bacteria, nor has it been reported in the aspect of live anti-bacteria

Method used

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  • Alexin and application thereof to preparation of antibacterial medicament
  • Alexin and application thereof to preparation of antibacterial medicament
  • Alexin and application thereof to preparation of antibacterial medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1. Expression vector construction method of Chlorella ellipsoides nitrate reductase mutant

[0066] 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

[0067] Example 2. Using the pGreen0029 framework to construct an expression vector.

[0068] Design primers according to Nos terminator sequence (SEQ ID NO: 6), upstream primer: 5' ATAAGAATGCGGCCGC TCGAATTTCCCCGATCGTTCAAAC3' (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

[0069] Example 3. Construction of the expression vector pGreen-NR-U-mNP1 of Chlorella ellipsoides nitrate reductase mutant.

[0070] 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 ( Figure 4 ) 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 I and Not...

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Abstract

The invention expresses a refitted rabbit defensin NP-1 (mNP-1) by using chlorella. The defensin expressed in chlorella has a very strong inhibiting or killing effect on Gram-negative bacteria, Gram-positive bacteria and the like, and can be used for killing medicament-resistant bacteria. A result of the invention can be applied to preparation of a medicament which is resistant to the Gram-negative bacteria and the Gram-positive bacteria, and can be applied to preparation of medicament which is resistant to super bacteria.

Description

technical field [0001] The invention relates to a defensin and its application in the preparation of drugs against Gram-negative bacteria and positive bacteria. Specifically, it relates to defensin mNP-1 produced by Chlorella, and its application in the preparation of drugs against Gram-negative bacteria and positive bacteria. 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 it was named by Professor Lehrer of the University of Cal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/17A61K48/00A61P31/04
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孙永华杨鹤鸣张建中彭宜红张英涛
Owner 北京中加保罗生物科技有限公司
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