Anti-microbial fusion proteins and application thereof

A technology of fusion protein and antibacterial protein, which is applied in the field of biotechnology and genetic engineering, can solve the problems of antibacterial peptides with narrow antibacterial spectrum, easy to be metabolized out of the body, and unsatisfactory bactericidal effect, so as to enhance the bactericidal effect, improve the expression level, and prolong the antibacterial peptide in vivo. The effect of action time

Inactive Publication Date: 2018-01-19
重庆赛托斯创生物科技发展有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the current situation that the existing antimicrobial peptides have a narrow antibacterial spectrum and poor antibacterial effect, and at the same time, the bactericidal / permeability-increasing protein has a small molecular weight, is easily metabolized out of the body, and has unsatisfactory bactericidal effects. A recombinant vector containing the human Cathelicidin antibacterial peptide gene and the human bactericidal / permeability enhancing protein BPI active fragment gene. After transforming host cells, a more efficient and broad-spectrum recombinant antibacterial fusion protein is obtained

Method used

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  • Anti-microbial fusion proteins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of expression vector

[0038] According to the amino acid sequence of the antibacterial fusion protein and the preferred codon of Chinese hamster, the fusion protein gene was designed and synthesized, and the synthetic gene was cloned into the pLVX-Tight-Puro vector to construct the fusion protein expression vector. Finally, two forms of fusion protein gene expression vectors, BPI-LL37 or LL37-BPI21, were obtained. The difference between the two expression vectors is that the order of fusion is different, and the signal peptide of each protein located at the N' end guides the exocrine expression of the entire fusion protein. The schematic diagram of the construction of the vector is as follows figure 1 as shown, figure 1 A is the schematic diagram of the vector structure of pLVX-rBPI21-LL-37, figure 1 B is the structural representation of pLVX-LL-37-rBPI21 vector.

Embodiment 2

[0039] Example 2 Protein expression of expression vector

[0040] (1) The antibacterial peptide LL-37 and its signal peptide protein coding gene sequence, the GGSGG flexible peptide coding gene sequence, rBPI21 and its signal peptide protein coding gene sequence were obtained by artificial synthesis; at the same time, in order to enhance the expression level of the fusion protein, According to the preferred codon theory, the rare codons on the LL-37 gene and rBPI21 gene were replaced with the preferred codons of Chinese hamsters to optimize the gene sequence of the fusion protein. The encoded nucleotide sequences of optimized LL-37 and rBPI21 are respectively shown in SEQ ID NO: 10 and SEQ ID NO: 8

[0041] (2) Carry out single and double enzyme digestion identification on the constructed vector

[0042] When we designed and constructed the pLVX-LL-37-rBPI21 and pLVX-rBPI21-LL-37 vectors, we added BamHI and MluI restriction sites at the 5' end and 3' end of the target gene re...

Embodiment 3

[0046] Example 3 Exocrine expression of fusion protein in CHO cells

[0047] (1) Cell culture

[0048] Chinese hamster ovary cells (CHO cells) were cultured in DMEM / F12 medium containing 10% FBS at 37°C, 5% CO 2 Conditioned culture, cells grow to 70%-80% density, ready for transfection.

[0049] (2) Cell transfection by liposome method

[0050] Replace with fresh medium 1 hour before transfection. Referring to Table 2 below, dilute plasmid DNA and liposome transfection reagent in two centrifuge tubes with serum-free DMEM / F12 medium. Gently invert the centrifuge tube 3-4 times to mix the liquid.

[0051] Immediately add the diluted liposomes to the diluted plasmid DNA solution, vortex immediately or invert the centrifuge tube 3-4 times to mix the liquid evenly. Stand at room temperature for 20 minutes to form the liposome / DNA complex, and the standing time should not exceed 30 minutes. Add all the liposome / DNA complexes into the cell culture medium, shake the culture dish...

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Abstract

The invention belongs to the field of biotechnological genetic engineering, and mainly relates to construction of an expression vector of anti-microbial fusion proteins and application thereof. The construction aims at the problems of narrow antimicrobial spectrum and bad antimicrobial effects of antimicrobial peptides in the prior art, the anti-microbial fusion proteins utilize codon preference,a fusion protein gene is designed and synthesized, a recombined vector is constructed, and after the vector is introduced into host cells, and the fusion proteins containing an antimicrobial protein segment rBPI21 and an antimicrobial peptide LL-37 are obtained. The fusion proteins have large molecular weight, can prolong internal action time of fusion proteins, have wider antimicrobial spectrum and largely enhanced sterilization effects, can be widely applied to the anti-microbial, anti-endotoxin and other technology fields, and provide a new selection for biological sterilization medicaments.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, and mainly relates to the construction and application of an antibacterial fusion protein expression vector. Background technique [0002] Antibiotics are widely used in the field of medical and health care, and have played an important role in the treatment of infectious diseases and saving the lives of patients. However, due to the irrational use of antibiotics caused by various factors, drug-resistant strains have gradually increased, leading to the phenomenon of multi-drug resistance, resulting in the inability to effectively control the infection of pathogenic bacteria. This problem is even more serious in ICUs with high incidence of sepsis. Current studies have shown that in the blood culture results of patients with severe sepsis, Gram-negative bacteria were isolated in 62% of patients, and Gram-positive bacteria were isolated in 47%. The composition of pathogenic microorg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00A61K38/17A61P31/04
Inventor 李战徐祥袁培淞宋昱庆马琳琳
Owner 重庆赛托斯创生物科技发展有限公司
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