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Mouse beta-alexin 1 recombinant plasmids, polypeptides, uses and preparations thereof

A technology of recombinant plasmids and defensins, which is applied in botany equipment and methods, biochemical equipment and methods, and medical preparations containing active ingredients, etc., can solve the problem of flu vaccine development speed, flu virus mutation, flu vaccine time-consuming, Influenza virus is easy to mutate and other problems, to achieve direct anti-influenza virus activity, shorten preparation time, and reduce costs

Inactive Publication Date: 2012-05-23
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Influenza viruses are prone to mutations, and the preparation of influenza vaccines is very time-consuming, and the development speed of influenza vaccines is far behind the pace of influenza virus mutations

Method used

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  • Mouse beta-alexin 1 recombinant plasmids, polypeptides, uses and preparations thereof
  • Mouse beta-alexin 1 recombinant plasmids, polypeptides, uses and preparations thereof
  • Mouse beta-alexin 1 recombinant plasmids, polypeptides, uses and preparations thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of recombinant mouse β-defensin 1 polypeptide (mBD-1)

[0042] 1. Obtain the mouse mBD-1 gene

[0043] (1) Use Trizol reagent (purchased from Invitrogen) to extract mouse lung total RNA according to the following steps

[0044] 1) Take 100 mg of mouse lung tissue under sterile conditions.

[0045] 2) Add 1ml Trizol.

[0046] 3) Homogenate (should be thorough, then transfer to EP tubes, when tissue homogenate volume > 100mg, pack separately, 1ml / each EP tube).

[0047] 4) Mix by inverting for 10 times, and let stand at room temperature for 5 minutes.

[0048] 5) Add 1 / 5 volume (0.2ml) of chloroform (must be 1 / 5 of the total volume).

[0049] 6) Mix by inverting for 10 times, and let stand at room temperature for 5 minutes.

[0050] 7) Centrifuge at 12000 g for 15 min at 4°C.

[0051] 8) Transfer the upper aqueous phase (about 400 μl) to another 1.5ml EP tube.

[0052] 9) Add an equal volume of isopropanol (about 400 μl), mix well and let ...

Embodiment 2

[0116] Embodiment 2: Construction of mouse β-defensin 1 (mBD-1) eukaryotic recombinant plasmid

[0117] 1. Obtain the mouse mBD-1 gene

[0118] The operation is the same as in Example 1. The difference from Example 1 is the PCR amplification primers and enzyme cutting sites.

[0119] Upstream primer: 5'-GCG GAA TTC ATG AAA ACT CAT TAC TTT CTC CTG G-3’

[0120] (EcoR I restriction site and protective base are provided at the 5' end)

[0121] Downstream primer: 5'-GCG CTC GAG TCA GCT CTT ACA ACA GTT GGG CT-3’

[0122] (The 5' end is equipped with an Xho I restriction site plus a protective base, including a stop codon TGA)

[0123] 2. Pretreatment of eukaryotic vectors

[0124] The eukaryotic vector is pCDNA3.1(+) (purchased from Invitrogen)

[0125] (1) Digest the eukaryotic vector pcDNA3.1(+) with restriction enzymes EcoR I and Xho I, and the reaction volume is 20 μl (containing 1 μl of EcoR I enzyme, 1 μl of Xho I enzyme, and 10×M buffer 2 2μl, pcDNA3.1(+) eukar...

Embodiment 3

[0143] Embodiment 3: Anti-influenza virus experiment in vitro

[0144] 1. Determination of influenza virus TCID50:

[0145] A / PR / 8 / 34 strain (H1N1) was selected as the influenza virus, provided by the National Influenza Center, Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention.

[0146] (1) Before virus infection, the MDCK cells in the culture flask were digested with 0.25% trypsin, divided into 24-well plates, and cultured into monolayer cells with growth medium containing 10% calf serum and antibiotics.

[0147] (2) The prepared influenza virus suspension was serially diluted 10 times with sterile PBS to prepare for infecting MDCK cells.

[0148] (3) Aspirate the growth solution in the 24-well plate, wash it twice with sterile PBS, and try to remove bovine serum, because bovine serum contains non-specific inhibin of influenza virus, which will affect the replication of influenza virus.

[0149] (4) Drop the diluted virus o...

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Abstract

The invention constructs prokaryotic recombinant plasmid and eukaryotic recombinant plasmid of mouse Beta-defensin-1-polypeptide (mBD-1) by the gene engineering technology, leads the prokaryotic recombinant plasmid to transfer into engineering bacteria to carry out induction to effective expression mouse Beta-defensin-1-polypeptide (mBD-1) and carry out the extraction and purification of expression products and enzyme cutting of enterokinase so as to release mBD-1 which is mature and has activity; by experiment, the invention proves that the mBD-1 polypeptide and MDCK cell strain which can transfect the eukaryotic recombinant plasmid stably has anti-influenza virus function so as to develop a novel drug used for curing or preventing influenza.

Description

technical field [0001] The invention relates to a recombinant plasmid and polypeptide of recombinant mouse β-defensin 1 and its use and preparation method. Background technique [0002] β-defensins are a class of cationic polypeptides widely found in animals with a wide range of antimicrobial activities in recent years. They are mainly distributed in epithelial cells, including skin, respiratory tract, urinary tract, intestinal and oral mucosal epithelial cells, etc. Through genomics analysis, 29 genes encoding β-defensins (DEFB) have been discovered, human β-defensins (hBD) 1-5, 6 and mouse β-defensins (mBD) 1-8 have been cloned, 12, 29. The study found that the upstream sequence of the mouse β-defensin 1 gene (mBD-1) (from GenBank, #AA065510) lacks the binding sites of NF-κB, IL-6 and STAT, etc., and cannot be induced by LPS and pro-inflammatory cytokines Expression belongs to intrinsic expression. At present, the preparation of β-defensins at home and abroad is mainly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/74C12N15/79A61K38/17A61K48/00A61P31/16C12N15/12C07K14/47
Inventor 李明远王月玲李婉宜江滟巩天祥杨远王保宁李虹李燕杨伟民冯艳
Owner SICHUAN UNIV
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