Preparation method of fibroin fluorescent probe

Abstract

The invention discloses a preparation method of a fibroin fluorescent probe. The method comprises the following steps: screening a phage library by using a fibroin material to obtain a phage specifically bonded with fibroin proteins; carrying out gene sequencing; using an amino acid sequence corresponding to the gene obtained by sequencing as a fibroin affinity peptide primary sequence; carrying out polypeptide synthesis on the fibroin affinity peptide primary sequence by adopting a polypeptide synthesis technology to obtain fibroin affinity peptide powder; and bonding the fibroin affinity peptide powder with fluorescein to obtain a fibroin fluorescent probe. The phage screened by the invention has very high affinity to the fibroin material, is high in probe sensitivity, high in detection speed and wide in application prospect, and provides more choices for high-efficient targeting and identification of the fibroin protein and the fibroin material.

Description

technical field

[0001] The invention relates to a preparation method of silk fibroin fluorescent probe, which belongs to the field of biotechnology. Background technique

[0002] Biomacromolecules such as proteins and peptides are biocompatible, functional and diverse, and are important components of natural organisms, as well as necessary raw materials for humans to transform nature and improve the quality of life. Therefore, the qualitative and quantitative analysis of proteins in materials plays an extremely important role, and biological methods with high sensitivity are often used. The traditional methods for identification and quantification of protein content include biuret method, phenol reagent method and ultraviolet absorption method, etc., but these traditional methods have the disadvantages of low sensitivity and non-specific determination. With the advancement of science and technology, mass spectrometry, especially time-of-flight mass spectrometry (MALDI-TOF) ...

Examples

Embodiment 1

[0046] 1) Use the phage peptide library produced by commercial NEB to carry out 5 rounds of screening on the silk membrane to obtain the phage bound to silk fibroin, the specific process is as follows;

[0047] 1.1) Preparation of silk film:

[0048]a. Obtain an aqueous silk protein solution with a concentration of 1 mg / mL;

[0049] b. Drop the fibroin solution on a polyethylene plate to air-dry, add alcohol to make it solidify, and obtain a water-insoluble silkworm fibroin film.

[0050] 1.2) Screening of polypeptide sequences bound to silk fibroin

[0051] a. Sealed silk fibroin material: add the buffer solution of TBS+BSA (0.5mg / ml) to the silkworm silk fibroin film;

[0052] b. Screening of phage library by silk fibroin material: For each phage species, add 10 microliters of the commercialized M13-12 phage random display library to the silk film and shake at 150rpm for 0.5h in a shaker at room temperature;

[0053] c. Washing: wash the silk fibroin film once with TBST w...

Embodiment 2

[0065] 1) Use the phage peptide library produced by commercial NEB to carry out 5 rounds of screening on the silk membrane to obtain the phage bound to silk fibroin, the specific process is as follows;

[0066] 1.1) Preparation of silk film:

[0067] a. Obtain an aqueous silk protein solution with a concentration of 10 mg / mL;

[0068] b. Drop the fibroin solution on a polyethylene plate to air-dry, add alcohol to make it solidify, and obtain a water-insoluble silkworm fibroin film.

[0069] 1.2) Screening of polypeptide sequences bound to silk fibroin

[0070] a. seal silk fibroin material: add the buffer solution of TBS+BSA (10mg / ml) in silkworm silk fibroin film;

[0071] b. Screening of phage library by silk fibroin material: for each phage species, 10 microliters of commercial M13-12 phage random display library was added to the silk film, and shaken at 220rpm for 0.5h in a shaker at room temperature;

[0072] c. Washing: wash the silk fibroin film 10 times with TBST wa...

Embodiment 3

[0084] 1) Use the phage peptide library produced by commercial NEB to carry out 5 rounds of screening on the silk membrane to obtain the phage bound to silk fibroin, the specific process is as follows;

[0085] 1.1) Preparation of silk film:

[0086] a. obtain silk protein aqueous solution, concentration is 50mg / mL;

[0087] b. Drop the fibroin solution on a polyethylene plate to air-dry, add alcohol to make it solidify, and obtain a water-insoluble silkworm fibroin film.

[0088] 1.2) Screening of polypeptide sequences bound to silk fibroin

[0089] a. Sealed silk fibroin material: add the buffer solution of TBS+BSA (1mg / ml) to the silkworm silk fibroin film;

[0090] b. Screening of phage library by silk fibroin material: for each phage species, 10 microliters of commercial M13-12 phage random display library was added to the silk film, and shaken at 220rpm for 0.5h in a shaker at room temperature;

[0091] c. Washing: wash the silk fibroin film 10 times with TBST washing...

technical field

[0001] The invention relates to a preparation method of silk fibroin fluorescent probe, which belongs to the field of biotechnology. Background technique

[0002] Biomacromolecules such as proteins and peptides are biocompatible, functional and diverse, and are important components of natural organisms, as well as necessary raw materials for humans to transform nature and improve the quality of life. Therefore, the qualitative and quantitative analysis of proteins in materials plays an extremely important role, and biological methods with high sensitivity are often used. The traditional methods for identification and quantification of protein content include biuret method, phenol reagent method and ultraviolet absorption method, etc., but these traditional methods have the disadvantages of low sensitivity and non-specific determination. With the advancement of science and technology, mass spectrometry, especially time-of-flight mass spectrometry (MALDI-TOF) ...

Examples

Embodiment 1

[0046] 1) Use the phage peptide library produced by commercial NEB to carry out 5 rounds of screening on the silk membrane to obtain the phage bound to silk fibroin, the specific process is as follows;

[0047] 1.1) Preparation of silk film:

[0048]a. Obtain an aqueous silk protein solution with a concentration of 1 mg / mL;

[0049] b. Drop the fibroin solution on a polyethylene plate to air-dry, add alcohol to make it solidify, and obtain a water-insoluble silkworm fibroin film.

[0050] 1.2) Screening of polypeptide sequences bound to silk fibroin

[0051] a. Sealed silk fibroin material: add the buffer solution of TBS+BSA (0.5mg / ml) to the silkworm silk fibroin film;

[0052] b. Screening of phage library by silk fibroin material: For each phage species, add 10 microliters of the commercialized M13-12 phage random display library to the silk film and shake at 150rpm for 0.5h in a shaker at room temperature;

[0053] c. Washing: wash the silk fibroin film once with TBST w...

Embodiment 2

[0065] 1) Use the phage peptide library produced by commercial NEB to carry out 5 rounds of screening on the silk membrane to obtain the phage bound to silk fibroin, the specific process is as follows;

[0066] 1.1) Preparation of silk film:

[0067] a. Obtain an aqueous silk protein solution with a concentration of 10 mg / mL;

[0068] b. Drop the fibroin solution on a polyethylene plate to air-dry, add alcohol to make it solidify, and obtain a water-insoluble silkworm fibroin film.

[0069] 1.2) Screening of polypeptide sequences bound to silk fibroin

[0070] a. seal silk fibroin material: add the buffer solution of TBS+BSA (10mg / ml) in silkworm silk fibroin film;

[0071] b. Screening of phage library by silk fibroin material: for each phage species, 10 microliters of commercial M13-12 phage random display library was added to the silk film, and shaken at 220rpm for 0.5h in a shaker at room temperature;

[0072] c. Washing: wash the silk fibroin film 10 times with TBST wa...

Embodiment 3

[0084] 1) Use the phage peptide library produced by commercial NEB to carry out 5 rounds of screening on the silk membrane to obtain the phage bound to silk fibroin, the specific process is as follows;

[0085] 1.1) Preparation of silk film:

[0086] a. obtain silk protein aqueous solution, concentration is 50mg / mL;

[0087] b. Drop the fibroin solution on a polyethylene plate to air-dry, add alcohol to make it solidify, and obtain a water-insoluble silkworm fibroin film.

[0088] 1.2) Screening of polypeptide sequences bound to silk fibroin

[0089] a. Sealed silk fibroin material: add the buffer solution of TBS+BSA (1mg / ml) to the silkworm silk fibroin film;

[0090] b. Screening of phage library by silk fibroin material: for each phage species, 10 microliters of commercial M13-12 phage random display library was added to the silk film, and shaken at 220rpm for 0.5h in a shaker at room temperature;

[0091] c. Washing: wash the silk fibroin film 10 times with TBST washing...

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