Disulfide bond protein based on Gluc luciferase and preparation method

Abstract

The invention discloses disulfide bond protein based on Gluc luciferase and a preparation method. The method comprises the following steps: introducing SEP-tag to the terminal C of Gluc luciferase gene; inserting the Gluc luciferase gene connected with an SEP tag into a multi-cloning site of an expression vector to obtain a luciferase Gluc-SEP gene expression vector; transforming the expression vector into an escherichia coli strain with trxB<-> / gor<-> resistance gene and DsbC redox gene to obtain an expression strain containing the expression vector; and cultivating the expression strain, and expressing to obtain disulfide bond protein based on Gluc luciferase. The method solves the problems of low optical stability of luciferase and quick fluorescence quenching.

Description

technical field

[0001] The invention relates to the technical field of protein preparation, in particular to a Gluc luciferase-based disulfide bond protein and a preparation method. Background technique

[0002] Medical imaging technology is an important method to explore the subtle life in the body. It can realize the highly targeted treatment of the occurrence and growth of diseases in the living body at the molecular level, and it is a new health care model. Medical imaging technologies mainly include nuclear magnetic resonance MRI, CT, medical ultrasound and optical imaging. Bioluminescence imaging (Bioluminescence imaging) is an important imaging method in optical imaging. It has the advantages of no need for light source excitation, no scattering, high transmittance, extremely low background interference and high signal-to-noise ratio. Under aerobic conditions, luciferase is used as a reporter source in vivo, and it emits light by interacting with a substrate. In rec...

Examples

Embodiment 1

[0040] Construction of Luciferase Gluc-SEP Gene Expression Vector

[0041] 1. Primers

[0042] Forward primer:

[0043] 5'-CGCGGATCCATGAAGCCCACCGAGAACAACGAA-3'; (SEQ ID NO: 1)

[0044] Reverse primer:

[0045]5'-ACGCCGTCGACGTCGTCGTCTCCGTCGTCGTCTCCGTCACCACCGGCCCCCTTGATCTT-3' (SEQ ID NO: 2)

[0046] 2. PCR amplification of Gluc luciferase gene

[0047] PCR reaction system:

[0048] 5μL 10×High Figelity PCR buffer, 15mM MgCl 2 , 1mM dNTP Mix, 0.3μM forward primer, 0.3μM reverse primer, 10pg template, 1.25U High Fidelity PCR Enzyme Mix, and finally add double distilled water to make the total system 50μL;

[0049] PCR reaction conditions:

[0050] Pre-denaturation at 94°C for 3min, denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, reaction cycle 25 times, and final extension at 72°C for 10min.

[0051] 3. Construction of luciferase Gluc-SEP gene expression vector

[0052] The PCR amplification product containing the Gluc luciferase gene o...

Embodiment 2

[0058] Preparation of Gluc-SEP-Shuffle expression strain

[0059] The luciferase Gluc-SEP gene expression vector of Example 1 was transformed into Escherichia coli competent cell DH5α by heat treatment at 42°C, recovered by culturing at 37°C and applied to LB plate (containing 25 μg / ml kanamycin) for screening, Colonies were picked for colony PCR and double enzyme digestion to identify positive clones. The positive strains screened were cultured, plasmids were extracted, and finally identified by gene sequencing; after that, the correct plasmids identified by sequencing were heat-treated and transformed into SHuffle T7B E.coli expression strains, and Glycerol bacteria were preserved and frozen for future use.

Embodiment 3

[0061] Expression and purification of Gluc-SEP-Shuffle protein

[0062] First, inoculate the frozen Glycerolbacterium Gluc-SEP-SHuffle into LB liquid medium (containing 25 μg / ml ampicillin), cultivate the activated strain overnight at 37°C and 225 rpm, and then inoculate the activated bacterial liquid at a ratio of 1:100 In fresh LB liquid medium (containing 100 μg / ml ampicillin), cultivate until OD600 reaches 0.4-0.6, which is the growth logarithmic phase of the strain, and then add the inducer isopropyl-β-D-1-sulfur The galactoside IPTG (final concentration: 1mM) induced protein expression at 37°C for 7h, centrifuged at 11000rpm at 4°C for 10min to collect the bacteria, discarded the supernatant LB medium, added a certain amount of binding buffer (20mM Na 3 PO 4 , 0.5M NaCl, 40mM imidazole solution, pH 7.4) suspend the bacteria, use ultrasonic to break the bacteria, ultrasonic 3sec, interval 4sec, ultrasonic until the solution is completely clear and transparent, then centr...

technical field

[0001] The invention relates to the technical field of protein preparation, in particular to a Gluc luciferase-based disulfide bond protein and a preparation method. Background technique

[0002] Medical imaging technology is an important method to explore the subtle life in the body. It can realize the highly targeted treatment of the occurrence and growth of diseases in the living body at the molecular level, and it is a new health care model. Medical imaging technologies mainly include nuclear magnetic resonance MRI, CT, medical ultrasound and optical imaging. Bioluminescence imaging (Bioluminescence imaging) is an important imaging method in optical imaging. It has the advantages of no need for light source excitation, no scattering, high transmittance, extremely low background interference and high signal-to-noise ratio. Under aerobic conditions, luciferase is used as a reporter source in vivo, and it emits light by interacting with a substrate. In rec...

Examples

Embodiment 1

[0040] Construction of Luciferase Gluc-SEP Gene Expression Vector

[0041] 1. Primers

[0042] Forward primer:

[0043] 5'-CGCGGATCCATGAAGCCCACCGAGAACAACGAA-3'; (SEQ ID NO: 1)

[0044] Reverse primer:

[0045]5'-ACGCCGTCGACGTCGTCGTCTCCGTCGTCGTCTCCGTCACCACCGGCCCCCTTGATCTT-3' (SEQ ID NO: 2)

[0046] 2. PCR amplification of Gluc luciferase gene

[0047] PCR reaction system:

[0048] 5μL 10×High Figelity PCR buffer, 15mM MgCl 2 , 1mM dNTP Mix, 0.3μM forward primer, 0.3μM reverse primer, 10pg template, 1.25U High Fidelity PCR Enzyme Mix, and finally add double distilled water to make the total system 50μL;

[0049] PCR reaction conditions:

[0050] Pre-denaturation at 94°C for 3min, denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, reaction cycle 25 times, and final extension at 72°C for 10min.

[0051] 3. Construction of luciferase Gluc-SEP gene expression vector

[0052] The PCR amplification product containing the Gluc luciferase gene o...

Embodiment 2

[0058] Preparation of Gluc-SEP-Shuffle expression strain

[0059] The luciferase Gluc-SEP gene expression vector of Example 1 was transformed into Escherichia coli competent cell DH5α by heat treatment at 42°C, recovered by culturing at 37°C and applied to LB plate (containing 25 μg / ml kanamycin) for screening, Colonies were picked for colony PCR and double enzyme digestion to identify positive clones. The positive strains screened were cultured, plasmids were extracted, and finally identified by gene sequencing; after that, the correct plasmids identified by sequencing were heat-treated and transformed into SHuffle T7B E.coli expression strains, and Glycerol bacteria were preserved and frozen for future use.

Embodiment 3

[0061] Expression and purification of Gluc-SEP-Shuffle protein

[0062] First, inoculate the frozen Glycerolbacterium Gluc-SEP-SHuffle into LB liquid medium (containing 25 μg / ml ampicillin), cultivate the activated strain overnight at 37°C and 225 rpm, and then inoculate the activated bacterial liquid at a ratio of 1:100 In fresh LB liquid medium (containing 100 μg / ml ampicillin), cultivate until OD600 reaches 0.4-0.6, which is the growth logarithmic phase of the strain, and then add the inducer isopropyl-β-D-1-sulfur The galactoside IPTG (final concentration: 1mM) induced protein expression at 37°C for 7h, centrifuged at 11000rpm at 4°C for 10min to collect the bacteria, discarded the supernatant LB medium, added a certain amount of binding buffer (20mM Na 3 PO 4 , 0.5M NaCl, 40mM imidazole solution, pH 7.4) suspend the bacteria, use ultrasonic to break the bacteria, ultrasonic 3sec, interval 4sec, ultrasonic until the solution is completely clear and transparent, then centr...

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