Preparation and application of novel swine-origin fusion antimicrobial peptide VASP biological preparation

A biological material and animal technology, applied in the field of biotechnology and genetic engineering, can solve the problems of low biological activity, narrow antibacterial spectrum, high cost of chemical synthesis, etc., and achieve the effect of promoting expression, improving survival rate, and improving immune and anti-infection ability

Inactive Publication Date: 2018-11-06
深圳市前海金卓生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The molecular weight of antimicrobial peptides is small, and there are certain difficulties in separation, purification and inspection
At present, the main source of antimicrobial peptides in scientific research and application is chemical synthesis, but the cost of chemical synthesis is high and the quantity is small, which is not conducive to large-scale application
Moreover, the existing single antimicrobial peptide molecule has disadvantages such as narrow antimicrobial spectrum and low biological activity; by using genetic engineering means, multiple antimicrobial peptide genes are recombined to obtain a broad-spectrum antimicrobial (G+, G-, virus, Fungi, parasites) and highly efficient recombinant antimicrobial peptide molecules with immunomodulatory activity are an important new way to overcome the defects of existing technologies and products
[0006] Reports on the fusion cloning and expression of antimicrobial peptide genes and other related genes are gradually increasing, but there are few reports on the fusion of short peptide genes with various antibacterial and immunomodulatory activities, and effective co-expression to form a variety of anti-infective active molecules and synergistic Anti-infection and Improving Immunity Level Reports

Method used

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  • Preparation and application of novel swine-origin fusion antimicrobial peptide VASP biological preparation
  • Preparation and application of novel swine-origin fusion antimicrobial peptide VASP biological preparation
  • Preparation and application of novel swine-origin fusion antimicrobial peptide VASP biological preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, porcine antimicrobial peptide fusion protein (VASP) has antibacterial effect

[0093] The porcine antimicrobial peptide fusion protein used in the present invention is named VASP, and its amino acid sequence with TPA secretion signal peptide is shown in sequence 1 in the sequence listing, and the nucleotide sequence of the gene VASP encoding protein VASP is shown in sequence 2.

[0094] Among them, the 1st-25th position of the sequence 1 is the TPA secretion signal peptide, the 26th-160th position is the porcine fusion antimicrobial peptide, and the 161-166th is the tag molecule. The 1st-75th position of the sequence 2 is the TPA secretion signal peptide encoding gene, the 76th-480th position is the porcine fusion antimicrobial peptide encoding gene, and the 481-498th is the encoding tag molecular gene.

[0095] 1. Preparation of eukaryotic expression vector pVAX1-SP

[0096] Replace the DNA fragment between the Hind III and Xbal recognition sequences of ...

Embodiment 2

[0162] Example 2, Research on biological activity of fusion gene recombinant eukaryotic expression plasmid in mice

[0163] 1. Detection of endotoxin

[0164] Use the "gel method" to detect the endotoxin content of the plasmid. For the method, refer to the "Instructions for Limulus Reagent". The specific method is as follows:

[0165] (1) Positive control sample preparation: take endotoxin standard substance (Shanghai Haring Biotechnology Co., Ltd., 150600), and prepare a solution with a concentration of 2 times sensitivity λ=0.25Eu / ml with water for inspection;

[0166] (2) Negative control: check water;

[0167] (3) Test sample: According to the test sample endotoxin limit value, the effective dilution factor of the calculator (MVD=cL / λ) is required, and the sample is diluted for later use;

[0168] (4) Take the LAL reagent, break off the neck of the ampoule and mark it, 1 of which is used as a positive control tube, 1 is used as a negative control tube, 2 are used as a te...

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Abstract

The invention discloses preparation and application of a novel swine-origin fusion antimicrobial peptide VASP biological preparation. The fusion swine antimicrobial peptide VASP is selected from one of the following proteins (A1), (A2) or (A3): (A1) protein with an amino acid sequence being a sequence 1 in a sequence table; (A2) a protein obtained by substituting and / or deleting and / or adding oneor several amino acid residues of an amino acid sequence expressed by a sequence 2 in the sequence table and provided with the same function; and (A3) a fusion protein obtained by connecting a tag with an N terminal and / or a C terminal of (A1) or (A2). Experiments show that VASP is capable of accelerating lymphocyte proliferation and increase of the number of leukocytes, inhibiting the growth of pathogenic microorganisms, enhancing humoral immunity (IgG, IgG1 and IgG2a) and cellular immunity (CD4+, CD8+T cells), and improving the immunoregulation capability and the anti-infection survival rateof animals.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to the preparation and application of a novel porcine-derived fusion antimicrobial peptide VASP biological preparation. Background technique [0002] The main problems that restrict the further development of my country's breeding industry and improve economic benefits are the prevention and control of animal diseases, the improvement of animal product quality and the development and production of high-quality and cheap feed. my country's export of animal products accounts for a very low proportion due to quarantine and drug residue problems. For example, pork is more than 230,000 tons, accounting for only about 4% of the international trade volume. In particular, 90% of protein raw materials such as soybean meal and 70% of energy feed raw materials such as corn in Sichuan animal feed depend on external supply, which seriously restricts the development of animal hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61P31/00A61P33/00A61P37/04
CPCA61K38/00A61P31/00A61P33/00A61P37/04C07K14/47C07K2319/036
Inventor 高荣彭俊杰魏泓万小平肖永乐陈建林朱玉华刘建华吕学斌王泽洲李江淩
Owner 深圳市前海金卓生物技术有限公司
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