Confluent protein capable of self-cracking into polypeptide with antibiosis and reparation function

A technology of fusion protein and peptide protein, applied in the direction of peptide/protein components, antifungal agents, local antibacterial agents, etc., can solve the problems of difficulty in obtaining antimicrobial peptides and insect antimicrobial peptides

Inactive Publication Date: 2004-06-16
BIOPHARM RES & DEV CENT JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] At present, it is difficult to mass-produce insect antimicrobial peptides by genetic engineering methods. The main problems are: 1. Antimicrobial peptide extraction and purification

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Expression of fusion protein of cecropin and acidic fibroblast growth factor

[0026]Primers F1 (SEQ ID NO: 22), F2 (SEQ ID NO: 23), R1 (SEQ ID NO: 24) and R2 (SEQ ID NO: 25) were designed and synthesized. The two primers F1 and R1 contain different DNA restriction endonuclease cutting sites, XhoI and XbaI respectively. The 5' end of F1 has a signal sequence behind the restriction endonuclease site to guide the secretion of the fusion protein in the host cell and excise it after translation. The 5' end of primer F2 and the 3' end of R1 contain the nucleotide sequence of the thrombin protein cleavage site (SEQ ID NO: 26). Two pairs of primers (F1R2 and F2R1) were used to carry out PCR reaction with the plasmid containing cecropin gene and acidic fibroblast factor (1-154a.a) gene as template, and the obtained product was used as template again, and primers F1 and R1 were used for PCR reaction. PCR reaction. The final PCR products were cloned and sequenced. ...

Embodiment 2

[0027] Example 2: Expression of fusion protein of cecropin and acidic fibroblast growth factor deletion variant (19-154a.a)

[0028] Primer F3 (SEQ ID NO: 27) was designed and synthesized, and its 3' end contained the nucleotide sequence of the thrombin protein cleavage site. Two pairs of primers (F1R2 and F3R1) were used to carry out PCR reaction with the plasmid containing cecropin gene and acidic fibroblast factor (19-154a.a) gene as template, and the obtained product was used as template again, and primers F1 and R1 were used for PCR reaction. PCR reaction. The final PCR products were cloned and sequenced. Digested with XhoI and XbaI and ligated into the pPICZαA vector generated by simultaneous digestion with XhoI and XbaI. Transformation into Pichia cells by electroporation followed by selection in YPD medium containing 100 μl / ml Zeocin resistance. Thus, clones stably expressing the fusion protein were obtained. Protein expression levels were determined by Western blo...

Embodiment 3

[0029] Example 3: Expression of fusion protein of cecropin and acidic fibroblast growth factor deletion variant (27-154a.a)

[0030] Primer F4 (SEQ ID NO: 28) was designed and synthesized, and its 3' end contained the nucleotide sequence of the thrombin protein cleavage site. Two pairs of primers (F1R2 and F4R1) were used to carry out PCR reaction with the plasmid containing cecropin gene and acidic fibroblast factor (27-154a.a) gene as template, and the obtained product was used as template again, and primers F1 and R1 were used for PCR reaction. PCR reaction. The final PCR products were cloned and sequenced. Digested with XhoI and XbaI and ligated into the pPICZαA vector generated by simultaneous digestion with XhoI and XbaI. Transformation into Pichia cells by electroporation followed by selection in YPD medium containing 100 μl / ml Zeocin resistance. Thus, clones stably expressing the fusion protein were obtained. Protein expression levels were determined by Western blo...

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PUM

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Abstract

The invention discloses an antibiotic peptide, blood coagulation apoenzyme slicing sequence and fusion protein sequence of phoroblast growth factor, wherein the antibiotic peptide can be bacteria resistant peptide, antimycotic peptide, or genetic engineering hybrid peptide for bacteria and fungus resistant simultaneously, while the phoroblast growth factor can be acidic phoroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF), the nucleic acid sequence for coding the fusion protein can be inserted into appropriate expression carrier and expressed in Pichia pastoris yeast.

Description

Technical field: [0001] The invention relates to the construction, development and application of a fusion protein composed of antibacterial peptide, thrombin protein cutting sequence and fibroblast growth factor. Background technique: [0002] The discovery of antibiotics is a milestone in the history of medicine, but with the extensive use of antibiotics, the resistance of pathogenic bacteria has also been greatly improved. What is more serious is that there are now antibiotic-resistant superbugs. Almost all antibiotics has no effect on them. Antimicrobial peptides can solve these problems. [0003] So far, humans have discovered and synthesized a large number of antimicrobial peptides, among which insect antimicrobial peptides are the representative. Experiments have shown that these antimicrobial peptides not only have broad-spectrum antibacterial ability against bacteria and fungi, but also have effects on viruses, protozoa and tumor cells. In addition, due to the un...

Claims

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Application Information

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IPC IPC(8): A61K38/18A61P17/02A61P31/02A61P31/10C07K19/00C12N15/62
Inventor 李校堃苏志坚郑青黄亚东吴晓萍许华何峰冯雅刘太胜
Owner BIOPHARM RES & DEV CENT JINAN
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