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A kind of cytoglobin-human lactoferrin peptide fusion protein, gene and application

A technology of fusion protein and lactoferrin, applied in the biological field, can solve problems such as unhelpful product functions, and achieve the effects of strong antibacterial effect, increased storage stability, and convenient purification.

Active Publication Date: 2021-12-03
QUANZHOU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fusion proteins used in these two methods do not contribute to the final product function, mainly to facilitate purification

Method used

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  • A kind of cytoglobin-human lactoferrin peptide fusion protein, gene and application
  • A kind of cytoglobin-human lactoferrin peptide fusion protein, gene and application
  • A kind of cytoglobin-human lactoferrin peptide fusion protein, gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Small amount of expression of TAT-CYGB-hLFcin in Escherichia coli

[0029] (1) Cell penetrating peptide TAT (SEQ NO.3), cytoglobin Cytoglobin (SEQ NO.4), and human lactoferrin peptide (SEQ NO.5) are sequentially connected in series to form cytoglobin-human lactoferrin peptide hybrid protein. The TAT-CYGB-hLFcin hybrid protein gene coding sequence was handed over to Gene Synthesis Company for whole gene synthesis through codon optimization software.

[0030] (2) Design full-length splicing primers to insert the TAT-CYGB-hLFcin gene into the expression vector pET32a(+) through the restriction sites Nde I and EcoR I ( figure 1 ), the accuracy of the final expression vector was confirmed by enzyme digestion and sequencing, and finally transferred to E. coli BL21 expression strains to obtain engineering strains containing TAT-CYGB-hLFcin fusion protein. Take 100 μL of the recombinant bacteria and evenly spread it on the LB plate (containing kanamycin sulfate at a...

Embodiment 2

[0032] Example 2 Separation and purification of TAT-CYGB-hLFcin fusion protein

[0033] (1) Collect the whole fermented bacteria containing supernatant, centrifuge to obtain the supernatant, resuspend in 20 mM Tris-HCl (pH 8.0) and centrifuge to obtain the protein supernatant, through cation exchange (CM Sepharose Fast Flow) chromatography, S -100 molecular sieve chromatography and Sephadex G-25 gel filtration chromatography desalted to obtain TAT-CYGB protein, SDS-PAGE electrophoresis detection, Western blotting qualitative inspection CYGB protein.

[0034] (2) CM cation exchange column: the column volume is 20mL, the UV detection wavelength is 220~280nm, and the column is equilibrated with 20mM Tris-HCl buffer (pH 8.0) at a flow rate of 1.5mL / min; The buffer was used to equilibrate the column again, and then the above buffer (pH 8.0) containing 500mM NaCl was used as the eluent for elution at a flow rate of 1.5mL / min. After collecting all the elution peak samples, all the c...

Embodiment 4

[0038] Example 4 Research on the Antibacterial Spectrum of TAT-CYGB-hLFcin Fusion Protein

[0039] (1) Detect the antibacterial activity of the fusion protein samples collected in step (5) of Example 2 against the strains listed in Table 1. Various bacteria were cultured in 5mL liquid medium shaker culture, and the culture conditions are listed in Table 1. When the bacterium grows to logarithmic growth phase, carry out the mensuration of inhibition zone according to the method of embodiment 1 method respectively. Measured 3 times to take the average value.

[0040] (2) Medium (g / L) composition. ① LB medium: tryptone 10g, yeast extract powder 5g, NaCl 10g, deionized water 1L, pH natural; ② Cha's medium: sucrose 30g, sodium nitrate 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, chlorine Potassium chloride 0.5g, ferrous sulfate 0.01g, deionized water 1L, pH natural; ③Nutritional agar: beef extract 3g, peptone 10g, NaCl 5g, agar 20g, deionized water 1L, pH 7.0; ...

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Abstract

The invention discloses a fusion protein of cytoglobin-human lactoferrin peptide, which is characterized in that the fusion protein is composed of transmembrane peptide (TAT), cell line protein (Cytoglobin, CYGB) and human lactoferrin peptide (human Lactoferricin, hLFcin) in series; use TAT to realize the secretion of the fusion protein to the outside of the cell for easy purification; use CYGB protein to drive the expression of lactoferrin peptide, and reduce the inhibition of antimicrobial peptides on host bacteria; the antioxidant properties of CYGB are beneficial to Increase the storage stability of antimicrobial peptides. The present invention can realize large-scale production of TAT‑CYGB‑hLFcin fusion protein, avoids the cumbersome steps of fusion protein design Linker, and simplifies the method of purifying the fusion protein by utilizing the stability characteristics of the fusion protein, and the fusion protein is shown to be used as an aquatic product preservation The prospects for application of additives in aquatic product preservation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cytoglobin-human lactoferrin peptide fusion protein, gene and application thereof. Background technique [0002] Aquatic products have occupied an important position in the daily diet of Chinese people, and people's preference for aquatic products has been further enhanced. Ensuring its fresh quality is not only to improve people's quality of life, but also to ensure people's food safety. Among them, aquatic products with long shelf life, soft and juicy, bright red color and delicious taste are popular among consumers and increasingly become the mainstream of aquatic product consumption. Since its processing and circulation at 0-4°C cannot completely inhibit the growth and reproduction of microorganisms, the quality of aquatic products changes mainly due to the large number of bacteria contained in them, such as Streptococcus, Pseudomonas, Bacillus, etc., causing Its s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A23B4/20
Inventor 段训威肖桂清杨槟煌陈洪彬董乐戴聪杰袁建军
Owner QUANZHOU NORMAL UNIV
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