CN107056899B

CN107056899B - Cell membrane positioning signal peptide and coding gene and application thereof

Info

Publication number: CN107056899B
Application number: CN201710398887.8A
Authority: CN
Inventors: 赵***; 孔祥东; 赵干业; 刘宁; 佘明聪; 代鹏; 赵勇江
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2017-05-31
Filing date: 2017-05-31
Publication date: 2020-07-17
Anticipated expiration: 2037-05-31
Also published as: CN107056899A

Abstract

The invention discloses a cell membrane positioning signal peptide, a coding gene and application thereof, wherein the signal peptide is (a) or (b); (a) a polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1; (b) a derivative polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 and has the function of a cell membrane positioning signal peptide. The invention adds 11 amino acid coding sequences on the basis of the DNA sequence of the coding Tat alkaline structural domain, and the formed cell membrane positioning signal peptide can target endogenous and exogenous target proteins to cell membranes. The cell membrane positioning signal peptide provided by the invention and a proper target protein form a fusion protein, and the fusion protein is added into a cell culture medium through intracellular overexpression or exogenous addition, so that the effectiveness of endogenous and exogenous protein cell membrane targeted positioning is enhanced, the fusion protein can be used for developing vaccines and medicines for treating virus, parasite and bacterial infection, and high-efficiency administration is realized.

Description

Cell membrane positioning signal peptide and coding gene and application thereof

Technical Field

The invention relates to a cell membrane positioning signal peptide, a coding gene and application thereof, belonging to the technical field of genetic engineering.

Background

Many human diseases, including neurodegenerative diseases, are currently incurable. The presence of cellular and tissue barriers, such as the blood-brain barrier (BBB) in certain cases of neurodegenerative diseases, prevents targeted transport of therapeutic molecules, reducing the ability of therapeutic molecules to reach their own targets. Increasing the biodistribution of tissue therapeutics by binding to cell-penetrating peptides (CPPs) that are capable of crossing biological membranes is a hot spot of research. CPPs are capable of carrying different therapeutic molecules, delivering them to their specific target and increasing their concentration in inaccessible tissues, thereby enhancing their therapeutic efficiency.

The polypeptide derived from the basic structural domain of the Tat protein of the HIV-1 transcription factor is the CPP which has the most prospect and is most clearly researched. It is expected to be used for treating various human diseases, particularly neurodegenerative diseases. It can be combined with different therapeutic molecules, including small molecules, antibodies, polypeptides, liposomes, nanoparticles, nucleic acids, and the like; which is effective in increasing the tissue permeability of the therapeutic agent and the efficiency of reaching biological targets.

Overexpression in the target cell after fusion of the DNA sequence encoding the basic domain of Tat with other protein genes drives the localization of the target protein in the nucleus. And the penetrating peptide formed by the Tat alkaline structural domain can enter cytoplasm through an endocytosis path after being fused with foreign protein, and is accumulated in the cytoplasm, and a small part of the penetrating peptide enters the nucleus. Therefore, the foreign protein can effectively act on targets in cytoplasm and nucleus. These properties of the Tat basic domain lay the foundation for the identification of novel protein transport signal peptides according to the invention. The protein transport signal peptide can drive the fusion protein to be positioned on the cell membrane, and the effectiveness of endogenous and exogenous protein cell membrane targeting positioning is enhanced.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a cell membrane localization signal peptide, a coding gene and applications thereof, wherein the cell membrane localization signal peptide can target endogenous (intracellular overexpression) and exogenous target proteins to cell membranes.

In order to achieve the purpose, the invention adopts the technical scheme that:

a cell membrane localization signal peptide, wherein the signal peptide is (a) or (b);

(a) a polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1;

(b) a derivative polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 and has the function of a cell membrane positioning signal peptide.

A gene sequence encoding said cell membrane localization signal peptide, said gene sequence being (a), (b) or (c);

(a) a nucleotide sequence shown as SEQ ID NO. 2;

(b) a nucleotide sequence which is hybridized with the nucleotide sequence shown in SEQ ID NO.2 under strict conditions and codes a protein with the function of a cell membrane positioning signal peptide;

(c) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.2 and codes a protein with the function of a cell membrane positioning signal peptide.

A recombinant fusion protein containing the cell membrane positioning signal peptide.

A recombinant expression vector, a recombinant bacterium or a transgenic cell line containing the gene sequence.

The application of the signal peptide in positioning the target protein in a cell membrane.

A method for localizing a target protein to a cell membrane, wherein the signal peptide is used as a cell membrane localization signal peptide to localize the target protein to the cell membrane.

The method for localizing the target protein to the cell membrane comprises the following steps:

(a) introducing the fusion gene into a target cell, thereby localizing the protein of interest on the cell membrane of the target cell; the fusion gene sequentially comprises a gene sequence for coding a signal peptide and a gene sequence for coding a target protein from upstream to downstream; or

(b) Adding the exogenous fusion protein into a target cell culture medium, so that the target protein crosses the cell membrane of the target cell, enters the target cell and is finally positioned on the cell membrane of the target cell; the exogenous fusion protein sequentially comprises a cell membrane positioning signal peptide and an amino acid sequence of a target protein from an N end to a C end.

The target cell is He L a cell.

The application of the cell membrane positioning signal peptide in preparing vaccines and medicines for treating virus, parasite and bacterial infection.

The invention has the beneficial effects that:

the invention adds 11 amino acid coding sequences on the basis of the DNA sequence of the coding Tat basic structural domain, and the formed cell membrane positioning signal peptide can target and position endogenous (intracellular overexpression) and exogenous target proteins on cell membranes. The cell membrane positioning signal peptide provided by the invention and a proper target protein form a fusion protein, and the fusion protein is added into a cell culture medium through intracellular overexpression or exogenous addition, so that the effectiveness of endogenous and exogenous protein cell membrane targeted positioning is enhanced, the fusion protein can be used for developing vaccines and medicines for treating virus, parasite and bacterial infection, and high-efficiency administration is realized.

Drawings

FIG. 1 is a vector map of recombinant plasmid pcDNA3.1-21aa-EGFP of the present invention.

FIG. 2 is a diagram showing the result of the verification of the construction of the recombinant plasmid pcDNA3.1-21aa-EGFP of the present invention, and

lanes

1 and 2 are both recombinant plasmids pcDNA3.1-21 aa-EGFP.

FIG. 3 is a map of the localization of the fusion protein expressed after transfection of He L a cells with recombinant plasmid pcDNA3.1-21aa-EGFP according to the present invention.

FIG. 4 is a vector map of recombinant plasmid pET-28a (+) -21aa-EGFP of the present invention.

FIG. 5 is a diagram showing the result of the verification of the construction of the recombinant plasmid pET-28a (+) -21aa-EGFP of the present invention, and

lanes

1 and 2 are both recombinant plasmids pET-28a (+) -21 aa-EGFP.

FIG. 6 is a map of the localization of the exogenous recombinant protein His-21aa-EGFP of the present invention in He L a cells.

Detailed Description

The following examples facilitate a better understanding of the invention, but do not limit the invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used, unless otherwise specified, were purchased from conventional biochemical stores.

Example 1 localization of overexpression of fusion proteins in He L a cells

Construction of eukaryotic recombinant expression plasmid

1. Design of primers

A PCR amplification primer which is synthesized by Shanghai Biotechnology Limited and contains a nucleotide sequence shown by SEQ ID NO.2 (a polypeptide consisting of 1 st to 21 st amino acid residues from the N end of SEQ ID NO.1 of an expression sequence table, which is called 21aa for short) specifically comprises the following components:

an upstream primer 21 For: 5' -CTGGTACCATGAAGTCGTGCTTCGCATGCCTAGTCTGCTTCATGGG ACGTAAGAAGCGACGACAGCGACGACGAGTGAGCAAGGGCGAGGAGC-3', the cleavage site for Kpn I (SEQ ID NO.3) is underlined;

the downstream primer 21 Rev: 5' -CTGAATTCTTACTTGTACAGCTCGTC-3', the EcoR I cleavage site (SEQ ID NO.4) is underlined.

2. PCR amplification, namely carrying out PCR amplification by taking the plasmid pEGFP-N1 as a template to obtain a PCR product with the size of 799bp, wherein a PCR amplification system (50 mu l) comprises 10 × buffer of 5 mu l and MgCl₂5. mu.l (25mM), 1. mu.l dNTP mix (10mM each), 0.5. mu.l Taq polymerase (5U/. mu.l), 1. mu.l 21For (10. mu.M), 1. mu.l 21Rev (10. mu.M), and 1. mu.l template, and ddH was added to the remaining₂And O. The PCR amplification conditions were: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 60 s; 10min at 72 ℃.

3. Enzyme digestion: the PCR product in step 2 was double digested with restriction enzymes Kpn I and EcoR I (digestion at 37 ℃ C. for 4h) to obtain a PCR digested product. Meanwhile, plasmid pcDNA3.1(+) is double digested with restriction enzymes Kpn I and EcoR I, and a linear plasmid DNA fragment of about 5400bp is recovered.

4. Connecting: and (3) connecting the PCR enzyme digestion product in the step (3) with the pcDNA3.1(+) linear DNA fragment to obtain a recombinant plasmid pcDNA3.1-21aa-EGFP, wherein the plasmid map of the pcDNA3.1-21aa-EGFP is shown in a figure 1. The recombinant plasmid pcDNA3.1-21aa-EGFP was double digested with Kpn I and EcoR I to obtain a 799bp DNA fragment as described in step 2 (FIG. 2). According to the sequencing result, the structural characteristics of the recombinant plasmid pcDNA3.1-21aa-EGFP are described as follows: a fusion fragment consisting of a gene sequence shown in SEQ ID NO.2 and a gene sequence coding green fluorescent protein EGFP is inserted between the Kpn I and EcoR I enzyme cutting sites of the plasmid pcDNA3.1(+), and a fusion protein of 21aa and EGFP, 21aa-EGFP for short is expressed.

II, positioning of fusion protein in He L a cell

The recombinant plasmid pcDNA3.1-21aa-EGFP is used for transfecting He L a cells, and the positioning of the fusion protein in the He L a cells is detected, wherein the specific method comprises the following steps:

1. culture of He L a cells by digesting cultured He L a cells into single cells with 0.25% pancreatin, and suspending the cells in DMEM medium containing 10% fetal calf serum to a cell concentration of 1.5 × 10⁵cells/ml, and then inoculated into 12-well cell culture plates, 1 ml/well, at 35 ℃ with 5% CO₂Culturing for 18-24h under the condition until the cells adhere to the wall, and preferably, the cell density is 70-80% during transfection.

2. Preparation of DNA/PEI mixtures: diluting every 1 μ g of recombinant plasmid pcDNA3.1-21aa-EGFP to 90 μ l with DMEM medium without serum and antibiotics; then adding PEI solution according to the proportion that 6 mul of 1mg/ml PEI (polyetherimide) solution is added into each 1 mul of recombinant plasmid, gently mixing evenly, and standing for 10min at room temperature.

3. Transfection: the old medium in the cell culture plate was discarded and 900. mu.l of fresh medium (DMEM medium containing 10% fetal bovine serum) was added to each well; adding the incubated DNA/PEI mixture to each well of the cell culture plate at a concentration of 1. mu.g recombinant plasmid per well, gently mixing, and adding 5% CO at 35 deg.C₂And (3) continuing culturing under the condition, marking cell nucleus by using Hoechst dye solution after 18-24h, observing the positioning of the fusion protein under a fluorescence microscope, wherein the result is shown in figure 3. the result of figure 3 shows that the fusion protein 21aa-EGFP is mainly positioned on the cell membrane of He L a cells.

Example 2 localization of exogenous fusion proteins in He L a cells

Construction of prokaryotic recombinant expression plasmid

1. Design of primers

an upstream primer 21' For:

5’-CTGGATCCATGAAGTCGTGCTTCGCATGCCTAGTCTGCTTCATGGGACGTAAGAAGCGACGACAGCGACGACGAGTGAGCAAGGGCGAGGAGC-3', the BamH I cleavage site (SEQ ID NO.5) is underlined;

downstream primer 21' Rev: 5' -GACTCGAGCTTGTACAGCTCGTC-3', Xho I cleavage sites are underlined (SEQ ID NO. 6).

2. PCR amplification, namely carrying out PCR amplification by taking the plasmid pEGFP-N1 as a template to obtain a PCR product with the size of 799bp, wherein a PCR amplification system (50 mu l) comprises 10 × buffer of 5 mu l and MgCl₂5. mu.l (25mM), 1. mu.l dNTP mix (10mM each), 0.5. mu.l Taq polymerase (5U/. mu.l), 1. mu.l 21 'For (10. mu.M), 1. mu.l 21' Rev (10. mu.M), 1. mu.l template, and ddH₂And O. The PCR amplification conditions were: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 60 s; 10min at 72 ℃.

3. Enzyme digestion: the PCR product in step 2 was double digested with restriction enzymes BamH I and Xho I (digestion at 37 ℃ C. for 4h) to obtain a PCR digested product. Meanwhile, plasmid pET-28a (+) was double-digested with restriction enzymes BamH I and Xho I, and a linear plasmid DNA fragment of about 5300bp was recovered.

4. And (2) connecting the PCR enzyme cutting product in the step (3) with a pET-28a (+) linear DNA fragment to obtain a prokaryotic expression recombinant plasmid pET-28a (+) -21aa-EGFP, and prokaryotic expression His-21aa-EGFP fusion protein, wherein a pET-28a (+) -21aa-EGFP plasmid map is shown in a figure 4. when BamH I and Xho I are used for double-enzyme cutting of the pET-28a (+) -21aa-EGFP plasmid, a 799bp DNA fragment (figure 5) in the step (2) is obtained, according to a sequencing result, a fusion fragment consisting of a gene sequence shown in SEQ ID NO.2 and a gene sequence coding green fluorescent protein EGFP is inserted between BamH I and Xho I enzyme cutting sites of the plasmid pET-28a (+), and the fusion protein of 21aa and EGFP with a 6 × label is expressed, and is called His-21 EGFP for short.

Secondly, expression and purification of fusion protein His-21aa-EGFP

1. Inducible expressionTransforming plasmid pET-28a (+) -21aa-EGFP into a strain E.coli B L21, picking out a single colony transformed by the corresponding plasmid after 12h, inoculating the single colony into 3-5ml L B culture medium (containing ampicillin 100 mu g/ml), and culturing at 37 ℃ for 12-18h, namely OD₆₀₀Stopping culturing when the ratio is more than 1.5, inoculating the bacterial liquid into 200ml L B culture medium (containing ampicillin 100 μ g/ml) at a volume ratio of 1:100, culturing at 37 deg.C for 2-3 hr until OD₆₀₀Is 0.6-0.8. 1ml of the bacterial solution was used as a control before induction, IPTG was added to the remaining bacterial solution to a final concentration of 0.1mM, and the mixture was transferred to 20 ℃ and cultured overnight at a low rotation speed. The next day, 1ml of the bacterial solution was taken as a control after induction, and the remaining bacterial solution was centrifuged at 6000rpm at 4 ℃ for 8min to collect the cells.

2. And (3) cracking thalli: the collected cells were resuspended in 20ml of phosphate-buffered saline (PBS) containing 140mM NaCl and 10mM Na₂HPO₄，2.7mM KCl，1.8mMKH₂PO₄pH 7.3, centrifuging the resuspension at 6000rpm for 8min at 4 ℃, discarding the supernatant, resuspending the washed thallus in 10ml of lysis buffer (20mM Tris-HCl, pH 7.4,100mM NaCl, 100mM imidozole, 0.1% Triton X-100, 1 × protease inhibitor) to prepare thallus suspension, performing ultrasonic disruption until the thallus suspension becomes clear to avoid generating a large amount of foam, supplementing PMSF (phenylmethylsulfonyl fluoride) to the final concentration of 1mM at proper time to reduce the degradation of protein, centrifuging at 14000rpm for 30min at 4 ℃, collecting the supernatant, and filtering with a 0.22 mu m-pore filter to remove impurities.

3. Affinity purification: 1ml of Ni was equilibrated beforehand with 5 column volumes of 5ml of equilibration buffer (20mM Tris-HCl, pH 7.4,100mM NaCl, 100mM azole) at room temperature²⁺sepharose magnetic beads. And adding the balanced magnetic beads into the cell lysis supernatant, incubating overnight at 4 ℃, and timely supplementing PMSF to a final concentration of 1mM so as to reduce the degradation of protein. The following day, the non-specifically adsorbed protein was eluted with 5ml of 5mM imidazole (dissolved in 20mM Tris-HCl, pH 7.4,500mM NaCl buffer). The target protein was eluted with 5ml of 50mM, 250mM, 500mM, imidazole (dissolved in 20mM Tris-HCl, pH 7.4,100mM NaCl buffer), respectively, and the three eluates were combined.

4. Protein dialysis and concentration: selecting a concentration tube with a filter membrane with a proper aperture according to the molecular weight of the protein, adding the eluent into the concentration tube, centrifuging at 4 ℃ and 5000rpm for 10-30min, concentrating to below 0.5ml, adding 10ml PBS, mixing uniformly, continuing to concentrate, and repeating for 2-3 times. Low temperature operation to prevent protein degradation.

5. Determination of protein concentration

Protein concentration was determined using the formula and corrected with the BSA standard. Protein concentration ═ a [ ("a₂₈₀×1.5-A₂₆₀× 0.75.75)/extinction coefficient]× dilution factor, wherein A₂₆₀And A₂₈₀The absorbance at 260nm and 280nm after dilution of the protein sample, respectively.

Thirdly, positioning of the fusion protein in He L a cells

1. Culture of He L a cells

Culture of He L a cells by digesting cultured He L a cells into single cells with 0.25% pancreatin, and suspending the cells in DMEM medium containing 10% fetal calf serum to a cell concentration of 1.5 × 10⁵cells/ml, and then inoculated into 12-well cell culture plates, 1 ml/well, at 35 ℃ with 5% CO₂Culturing for 18-24h under the condition.

2. Addition of foreign proteins

Discarding the old medium from the cell culture plate, and adding 900. mu.l of fresh medium to each well; adding the target protein to each well of the cell culture plate to a final concentration of 30nM, gently mixing, and adding 5% CO at 35 deg.C₂And after 12 hours, marking the cell nucleus by using Hoechst dye solution, observing the positioning of the fusion protein under a fluorescence microscope, wherein the result is shown in figure 6. the result of figure 6 shows that the fusion protein His-21aa-EGFP is mainly positioned on the cell membrane of He L a cells.

The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> first subsidiary Hospital of Zhengzhou university

<120> cell membrane localization signal peptide, and coding gene and application thereof

<160>6

<170>PatentIn version 3.5

<210>1

<211>21

<212>PRT

<213> HIV Tat basic Domain

<400>1

Lys Ser Cys Phe Ala Cys Leu Val Cys Phe Met Gly Arg Lys Lys Arg

1 5 10 15

Arg Gln Arg Arg Arg

20

<210>2

<211>63

<212>DNA

<213> HIV Tat basic Domain

<400>2

aagtcgtgct tcgcatgcct agtctgcttc atgggacgta agaagcgacg acagcgacga 60

cga 63

<210>3

<211>93

<212>DNA

<213> Artificial sequence

<400>3

ctggtaccat gaagtcgtgc ttcgcatgcc tagtctgctt catgggacgt aagaagcgac 60

gacagcgacg acgagtgagc aagggcgagg agc 93

<210>4

<211>26

<212>DNA

<213> Artificial sequence

<400>4

ctgaattctt acttgtacag ctcgtc 26

<210>5

<211>93

<212>DNA

<213> Artificial sequence

<400>5

ctggatccat gaagtcgtgc ttcgcatgcc tagtctgctt catgggacgt aagaagcgac 60

gacagcgacg acgagtgagc aagggcgaggagc 93

<210>6

<211>23

<212>DNA

<213> Artificial sequence

<400>6

gactcgagct tgtacagctc gtc 23

Claims

1. A cell membrane localization signal peptide, wherein said signal peptide is (a):

polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 1.

2. A gene sequence encoding the cell membrane localization signal peptide of claim 1, wherein the gene sequence is the nucleotide sequence shown in SEQ ID No. 2.

3. A recombinant fusion protein comprising the cell membrane localization signal peptide of claim 1.

4. A recombinant expression vector, recombinant bacterium or transgenic cell line comprising the gene sequence of claim 2.