Truncated body of Seroin protein and application of truncated body

A protein and sequence technology, applied in the application field of the protein truncation body, can solve the problems of incomplete folding, high synthesis cost, host toxicity or growth inhibition, etc., to reduce cost and difficulty, low production cost, shorten molecular weight Effect

Active Publication Date: 2021-02-19
SOUTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prokaryotic expression requires the use of bacteria such as Escherichia coli as expression strains, and seroin, as a type of protein with strong antibacterial activity, has an unavoidable problem in obtaining its recombinant protein through prokaryotic expression: seroin protein has a negative effect on the host. Toxic or growth inhibitory
This issue can result in strains that grow slowly or fail to express, or only express incompletely folded, functionally restricted recombinant proteins
Therefore, the method of obtaining seroin protein through prokaryotic expression has great uncertainty, and the production efficiency, product yield and product activity cannot be guaranteed
Chemical synthesis is currently mainly used to synthesize small molecular proteins less than 10kDa, but for proteins above 10kDa, the synthesis cost is high and the synthesis is difficult. Therefore, the study of biologically active seroin truncated fragments is of great significance for the development of Seroin antibacterial products

Method used

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  • Truncated body of Seroin protein and application of truncated body
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  • Truncated body of Seroin protein and application of truncated body

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1, seroin protein bioinformatics analysis

[0025] Nucleosides of seroin1 (gene number gi|19070653), seroin2 (gene number gi|19070655) and seroin3 (gene number gi|512931752) downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) The amino acid and amino acid sequences of seroin1, seroin2 and seroin3 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the nucleotide sequences of seroin1, seroin2 and seroin3 are shown in SEQ ID NO.4 , shown in SEQ ID NO.5 and SEQ ID NO.6. Then use the following online software and biological software for sequence analysis:

[0026] Protein signal peptide prediction: SignalP (http: / / www.cbs.dtu.dk / services / SignalP / );

[0027] Protein molecular weight and isoelectric point prediction: ExPASy (http: / / www.expasy.org / tools / );

[0028] Homology comparison software: Clustal X and GeneDoc;

[0029] The analysis results of amino acid sequence length, signal peptide, molecular weight and isoelectric point a...

Embodiment 2

[0034] Embodiment 2, seroin protein truncated synthesis

[0035] Based on previous research results, the three silkworm seroin proteins were divided into N-terminal and C-terminal parts after removing the signal peptide sequence, and the obtained sequences are shown in Table 2 (Dong et al., 2016). The full-length and truncated peptides of seroin protein were obtained by chemical synthesis, and they were named seroin1-N, seroin1-C, seroin2-N, seroin2-C, seroin3-N, and seroin3-C. The relevant information has been listed in Table 1. illustrate.

[0036] Table 2, the amino acid sequence of the truncated body of silkworm seroin protein

[0037]

Embodiment 3

[0038] Example 3, Verification of antibacterial activity of seroin protein truncated peptides

[0039]In order to evaluate the antibacterial activity of different seroin protein peptides, two kinds of bacteria were selected in this experiment: Escherichia coli (Gram-negative bacteria, G-) and Staphylococcus aureus (Staphylococcus aureus, Gram-positive bacteria, G+) were compared with Each seroin protein peptide segment was incubated, and the growth curve of the bacteria was detected by spectrophotometry, and the growth rate of the bacteria relative to the control group was calculated. The specific method is as follows:

[0040] 1) Using sterile phosphate buffer solution (PBS), dissolve a sufficient amount of Seroin protein peptides to a protein concentration of 1 mg / mL;

[0041] 2) Using LB liquid medium, culture the bacteria to OD 600 =0.2-0.3;

[0042] 3) Take 50 μL of protein solution and 150 μL of bacterial solution, add them to each well of a 96-well plate, mix thoroug...

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Abstract

The invention discloses a truncated body of a Seroin protein and application of the truncated body. The truncated body of the Seroin protein is an N-end or C-end fragment of Seroin 1, Seroin 2 or Seroin 3. The truncated polypeptide has antibacterial activity and can be used for resisting Gram-positive bacteria and Gram-negative bacteria, the molecular weight of protein is greatly reduced through truncation, and therefore the cost and difficulty of synthesizing the polypeptide by a chemical method are reduced, and compared with a previous complete protein production technology, the truncated body of a Seroin protein has the obvious advantages of low production cost, high activity, high stability, simplicity, convenience, quickness and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a truncated body of Seroin protein, and also relates to the application of the protein truncated body. Background technique [0002] The silkworm (Bombyx mori) is a typical representative of Lepidoptera insects. It originated in China and was domesticated from the original wild silkworm (Bombyx mandarina). It has important agricultural and economic significance (Sutherland et al., 2010). As a complete metamorphosis insect, the silkworm does not eat or exercise during the process of metamorphosis into a pupa, and only wraps itself in a cocoon to resist external harm. The silk that makes up the cocoon is an externally spun fibrin secretion. Silk is mainly composed of fibroin and sericin (Xiang Zhonghuai, 2005). Through mass spectrometry and other molecular biology research techniques to study and analyze the components of silk, it was found that besides silk fibroin and sericin, ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P31/04
CPCA61K38/00A61P31/04C07K14/43586Y02A50/30
Inventor 董照明赵萍夏庆友诸鸿韬
Owner SOUTHWEST UNIV
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