CN106046177B - P-5m-Fc fusion protein and its expression gene, preparation method and application - Google Patents

Abstract

The invention relates to a P-5m-Fc fusion protein, an expression gene, a preparation method and an application thereof, and belongs to the field of medical biotechnology. The P-5m-Fc fusion protein is formed by connecting a functional unit with the Fc part of human immunoglobulin IgG1, wherein the functional unit is formed by connecting the amino acid sequence shown in SEQ ID NO.1 and a linker, and the linker is A polypeptide consisting of six glycines connected in sequence, the P-5m-Fc fusion protein has at least one of the functional units, and when the P-5m-Fc fusion protein has a plurality of the functional units, more The functional units are connected in sequence and then connected to the Fc part of the immunoglobulin. The P-5m-Fc fusion protein fuses and expresses P-5m and the Fc fragment of human IgG1 into a peptibody, which can not only improve the efficacy of the P-5m octapeptide, but also significantly prolong the half-life of the drug, and obtain a good finished drug sex. And through experimental studies, it has been confirmed that it has a significant anti-tumor metastasis function.

Description

P-5m-Fc fusion protein and its expression gene, preparation method and application

technical field

The present invention relates to the technical field of medicine and biology, in particular to a P-5m-Fc fusion protein and its expression gene, preparation method and application.

Background technique

Current research shows that tumor metastasis is the root cause of death in cancer patients. The P-5m octapeptide is a polypeptide with eight amino acids derived from the fifth domain (D5) of high molecular kininogen, which is the shortest polypeptide that has been found to be derived from D5 and has an effective anti-tumor metastasis.

Studies have shown that P-5m octapeptide can inhibit the adhesion and invasion of mouse melanoma cells at the cellular level, and can inhibit the lung metastasis of mouse melanoma, and has the effect of inhibiting tumor cell metastasis in human HCCLM3 liver cancer cells. .

However, after administration of P-5m octapeptide, it has the defects of poor stability and short half-life, and it is still difficult to apply in clinical practice.

SUMMARY OF THE INVENTION

Based on this, it is necessary to provide a P-5m-Fc fusion protein for the above problems, which can prolong the stability and half-life of the P-5m octapeptide and enhance its druggability.

A P-5m-Fc fusion protein is formed by connecting a functional unit with the Fc part of human immunoglobulin IgG1, wherein the functional unit is formed by connecting the amino acid sequence shown in SEQ ID NO. The P-5m-Fc fusion protein is a polypeptide consisting of six glycines connected in sequence, and the P-5m-Fc fusion protein has at least one of the functional units, and when the P-5m-Fc fusion protein has a plurality of the functional units , a plurality of the functional units are connected in sequence and then connected with the Fc part of the immunoglobulin.

Preferably, the P-5m-Fc fusion protein includes two functional units.

The present invention also discloses an expression gene encoding the above-mentioned P-5m-Fc fusion protein, and the sequence of the expression gene is:

A) consisting of a combination of the nucleotide sequence of the functional unit and the nucleotide sequence of the Fc part encoding the immunoglobulin; or

B) a sequence that encodes a protein of the same sequence as the nucleotide sequence of A), but differs from the nucleotide sequence of A) due to the degeneracy of the genetic code; or

C) A nucleotide sequence in which one or more bases are substituted, deleted, or modified by addition of one or more bases to the nucleotide sequence shown in A) or B).

In one embodiment, in A), the nucleotide sequence of the functional unit is shown in SEQ ID NO.2.

The present invention also discloses an expression vector, which is an expression vector having the above-mentioned expression gene of the P-5m-Fc fusion protein.

Preferably, the expression vector is pET-28a(+)Fc plasmid.

The present invention also discloses a preparation method of the above-mentioned P-5m-Fc fusion protein, comprising the following steps:

(1) Constructing the above-mentioned expression gene, inserting the above-mentioned expression gene into a vector, and constructing a recombinant expression vector

(2) transferring the above-mentioned recombinant expression vector into a host cell, and obtaining a positive expression vector through screening;

(3) The above-mentioned positive expression vector is transferred into the expression cell, and the expression gene sequence in step (1) is induced to express, and that is, it is obtained.

In one embodiment, in step (1), a recombinant expression vector is constructed by the following method: first, a gene fragment whose base sequence composition is SEQ ID NO.2 is obtained, and pET-28a is double digested with BamH I and Nde I. (+) Fc plasmid, reclaims the vector fragment cut by enzyme, inserts the gene fragment of above-mentioned SEQ ID NO.2 into above-mentioned vector by DNA ligase, obtains recombinant plasmid, is recombinant expression vector;

In step (2), the positive expression vector is obtained by the following method: the ligation product is transferred into Escherichia coli competent DH5α by heat shock method, and coated on X-gal, isopropyl-β-D-thiohalogen On the LB agar medium of lactoside (IPTG) and ampicillin, a positive plasmid is obtained by screening, which is a positive expression vector;

In step (3), the specific method for inducing expression is: transfer the positive plasmid obtained in step (2) into E. coli E.coli BL21 (DE3) competent cells by heat shock method, and coat it on ampicillin-containing cells. On the LB agar medium of penicillin, culture; pick a single colony and inoculate it into LBG medium containing ampicillin, culture, add isopropyl-β-D-thiogalactoside after culture, and then induce culture; obtain P- 5m-Fc fusion protein.

In one embodiment, in step (2), the ligation product is transformed into E. coli competent DH5α by heat shock method, and the ligation product is coated with X-gal, isopropyl-β-D-thiogalactoside and On the LB agar medium of ampicillin, after culturing for 12-16 hours, a single colony was picked and inoculated into the LB medium containing 50 μg/ml ampicillin, and the positive plasmid was obtained by screening;

In step (3), the positive plasmid obtained in step (2) was transferred into Escherichia coli E.coli BL21 (DE3) competent cells by heat shock method, and coated on LB agar medium containing 50 μg/ml ampicillin cultured for 12-16h; pick a single colony and inoculate it into LBG medium containing 50μg/ml ampicillin, shake and culture until the OD ₆₀₀ reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside To the final concentration of 0.2-0.6 mmol/L, induce culture, centrifuge the culture, take the precipitate, add phosphate buffer and loading buffer, boil and centrifuge again to obtain the P-5m-Fc fusion protein.

In one embodiment, it also includes step (5) purification: collecting the induced-expressed thalline, suspending with phosphate buffer, sonicating, and centrifuging to obtain the P-5m-Fc fusion protein expressed in inclusion bodies; The above inclusion bodies were resuspended in lysis buffer, and then washed with Triton X/100 (polyethylene glycol octyl phenyl ether), sodium deoxycholate, and urea for preliminary purification. The preliminary purified inclusion bodies were washed with urea and DTT. (dithiothreitol) in Tris-HCl buffer solution for denaturation and dissolution, and then renaturation by redox agent or air oxidation under alkaline conditions to obtain an active P-5m-Fc fusion protein with correct conformation.

In the manner described above, the misfolded form of the protein present in the inclusion bodies can be refolded into an active P-5m-Fc fusion protein with the correct conformation.

In one embodiment, in the purification of the step (5): the above inclusion bodies are resuspended in a lysis buffer, the lysis buffer includes 50 mmol/L Tris-HCl at pH 8.0, 1 mmol/L Tris-HCl EDTA, 100mmol/1L NaCl, and then through the volume percentage of 2% Triton X/100, the mass percentage concentration of 0.2% sodium deoxycholate, 1mol/L urea washing for preliminary purification;

The preliminarily purified inclusion bodies were denatured and dissolved with 25 mmol/L Tris-HCl buffer containing 8 mmol/L urea and 20 mmol/L DTT at pH 8.0;

Then, the above-mentioned P-5m-Fc fusion protein is purified by SP-sepharose cation chromatography column, and then renatured by redox agent or air oxidation under alkaline conditions;

The conditions for renaturation of the redox agent are: in the purified P-5m-Fc fusion protein buffer, add DTT to a final concentration of 10 mmol/L, and then add reduced glutathione and oxidized glutathione To the concentration of 2mmol/L and 0.2mmol/L, respectively, dilute the P-5m-Fc fusion protein to 0.5mg/ml with 4mol/L urea, and gradually reduce the concentration from 4mol/L to 0mol/L urea buffer solution Dialysis renaturation, the urea solution contains the same concentration of reduced and oxidized glutathione;

The conditions of air oxidative renaturation under the described alkaline conditions are as follows: the purified P-5m-Fc fusion protein is dialyzed into a 25mmol/L Tris-HCl buffer of pH10.0 with a urea content of 8mol/L, and DTT is added. To the final concentration of 10 mmol/L, exposed to the air and stirred overnight, the P-5m-Fc fusion protein was diluted to 0.5 mg/ml, and the urea buffer solution whose concentration was gradually reduced from 4 mol/L to 0 mol/L was dialyzed and renatured;

An active P-5m-Fc fusion protein with the correct conformation was obtained by renaturation.

The refolding of the protein in the above manner has a good renaturation effect, and the purification by SP-sepharose cation chromatography column can further improve the purity of the fusion protein.

The invention also discloses the application of the above-mentioned P-5m-Fc fusion protein in the preparation of a medicine for inhibiting the metastasis of liver cancer cells.

In particular, it has a good inhibitory effect on the invasion of HCCLM3 cells into the matrix membrane, and can be used to inhibit the metastasis of human liver cancer HCCLM3 cells.

Compared with the prior art, the present invention has the following beneficial effects:

A P-5m-Fc fusion protein of the present invention, which fuses and expresses P-5m and the Fc fragment of human IgG1 into a peptibody, can not only improve the efficacy of the P-5m octapeptide, but also significantly prolong the half-life of the drug, Obtain good medicinal properties. And through experimental studies, it has been confirmed that it has a significant anti-tumor metastasis function.

The preparation method of the P-5m-Fc fusion protein of the present invention establishes the expression process of the P-5m-Fc fusion protein, and optimizes the process conditions therein.

In addition, the optimal screening of the renaturation conditions of the P-5m-Fc fusion protein expressed in inclusion bodies was carried out, and an active P-5m-Fc fusion protein that could refold the misfolded protein in the inclusion bodies into a correct conformation was obtained. - Optimal conditions for 5m-Fc fusion protein.

Description of drawings

FIG. 1 is a diagram showing the identification of double-enzyme digestion of pET-28a(+)P-5m-Fc plasmid in Example 1. FIG.

In the figure: 1 is the pET-28a(+)P-5m-Fc plasmid; 2 is the result of enzyme digestion; M is the DL2000 DNA Marker.

Figure 2 shows the expression of pET-28a(+)-Fc and pET-28a(+)P-5m-Fc in Example 2 without induction.

In the figure: 1 and 2 are the expression of pET-28a(+)-Fc without induction in E. coli, 3 and 4 are the expression of pET-28a(+)P-5m-Fc without induction in E. coli, M is Protein Marker, and the arrow points to the electrophoresis band of P-5m-Fc fusion protein.

Figure 3 shows the expression of pET-28a(+)-Fc and pET-28a(+)P-5m-Fc in Example 2 under induction.

In the figure: 1 is the expression of pET-28a(+)-Fc under induction in E. coli, 2 and 3 are the expression of pET-28a(+)P-5m-Fc under induction in E. coli, M is Protein Marker , the arrow points to the P-5m-Fc fusion protein electrophoresis band.

FIG. 4 shows the expression of pET-28a(+)P-5m-Fc inclusion bodies in Example 2. FIG.

In the figure: 1 and 2 are the pET-28a(+)P-5m-Fc expression proteins in the supernatant after lysis of the bacteria solution; 3 is the pET-28a(+) expression protein in the bacteria solution lysis precipitate; M is the Protein Marker, arrow Indicated is the P-5m-Fc fusion protein electrophoresis band.

FIG. 5 is the preliminary purification of the pET-28a(+)P-5m-Fc expression product in Example 2. FIG.

In the figure: 1, 2 and 3 are the pET-28a(+)P-5m-Fc expression protein in the supernatant after bacterial lysis; 4 and 5 are the pET-28a(+)P-5m-Fc protein after preliminary purification ; M is the Protein Marker, and the arrow points to the P-5m-Fc fusion protein electrophoresis band.

FIG. 6 shows the purification and Western-blotting results of the pET-28a(+)P-5m-Fc expression product in Example 2. FIG.

In the figure: 1 is the pET-28a(+)P-5m-Fc expression product after column purification, 2 is the Western-blotting result of pET-28a(+)P-5m-Fc, 3 is pET-28a(+ )-Western-blotting result of Fc, M is ProteinMarker.

FIG. 7 is a 400-fold magnification of the ability of P-5m-Fc to inhibit the invasion of HCCLM3 cells through transwell chambers in Example 3. FIG.

In the figure: Control represents blank control; 10 μmol of P-5m-Fc represents the result after treatment with P-5m-Fc with a final concentration of 10 μmol; 100 μmol of P-5m-Fc represents after treatment with P-5m-Fc with a final concentration of 100 μmol Results; P-5m-Fc100μmol+Ab indicates the result after co-treatment with P-5m-Fc and P-5m polyclonal antibody with a final concentration of 100μmol; P-5m10μmol indicates the result after treatment with P-5m with a final concentration of 10μmol; P-5m 100 μmol represents the result after treatment with a final concentration of 100 μmol of P-5m.

Figure 8 is a comparison of the ability of P-5m-Fc to inhibit the invasion of HCCLM3 cells through transwell chambers in Example 3.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully hereinafter with reference to the related drawings. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

Example 1

Construction of P-5m-Fc fusion protein expression vector

1. Methods.

1. Extraction and purification of bacterial plasmids

After analysis, the amino acid sequence of P-5m is GHGKHKNK. After screening through a large number of experiments, the inventor synthesized the positive and negative chains of the P-5m peptide gene using the codons preferred by Escherichia coli, adding ATG at the 5' end and 6 at the 3' end. glycine codons (the GGT GGT GGT GGT GGT GGT part below), and in order to increase the titer, the P-5m peptide gene and the glycine codon were repeated for two cycles. After annealing, the 5' and 3' ends were Generate Nde I recognition site (CAT ATG) and BamH I recognition site (GGA TCC): positive strand 5'-C CAT ATG GGC GAT GGC AAA GAT AAG AAC AAG GGT GGT GGT GGTGGT GGT GGC GAT GGC AAA GAT AAG AAC AAG GGA TCC GCG-3' (ie, the nucleotide sequence shown in SEQ ID NO. 2); 5'-CGC GGA TCC CTT GTT CTT ATC TTT GCC ATC GCC CAT ACC ACC ACC ACC ACC CTT GTT CTT ATC TTT GCC ATC GCC CAT ATG G-3'.

Then, according to the instructions provided by the Nde I and BamH I restriction enzyme kits (manufacturer: Promaga, models: R4024 and R6801), the enzyme digestion was performed, and pET-28a(+)Fc was double-digested with Nde I and BamH I enzymes. Plasmid (source: prepared by referring to the following documents: Fusion expression of TPO mimetic peptide and human IgG1 Fc fragment and study on its biological properties. Chinese Journal of Biological Engineering, 2002, 18(4): 424-430; wherein, the Plasmid for long Fc fragment: pCDNA3.1-hlgG1 Fc was purchased from Beijing Pubosi Cat. No. VIY0062hIgG1 Fc).

In the enzyme digestion step, single digestion is performed first, then DNA is extracted and precipitated, dissolved in TE, and then another digestion is performed. The digestion reaction was carried out according to the following system: 10 μL of PCR product recovered, 2 μL of 10×H buffer/3 μL of 10×T buffer, 1 μL of each of the two endonucleases, and finally added to 20 μL with ddH ₂ O, and digested in a water bath at 37°C for 1.5 h , 65 ℃ inactivated 15min. Identification by 0.8% agarose gel electrophoresis.

2. Rapid in-gel recovery of DNA by glass milk method

Agarose gel electrophoresis was performed on the above plasmid double-enzyme digestion products, 1.2% agarose gel electrophoresis was used to recover the annealed fragments containing P-5m and linkers, and 0.8% agarose gel electrophoresis was used to recover the digested vector fragments .

3. Annealing of DNA fragments

The positive and negative strands of the P-5m peptide gene were synthesized using the preferred codons of Escherichia coli, dissolved in sterile water to a concentration of 0.1 pmol/μL, mixed with the same amount of positive and negative strand oligonucleotides, and annealing buffer was added to 1x (10 mmol/LTris, pH 7.5-8.0, 50 mmol/L NaCl, 1 mmol/L EDTA), heat denatured at 95°C for 5 min, then slowly cooled to room temperature.

4. DNA sticky end ligation

T4 DNA ligase was used to connect the gene double strands of the above P-5m and the linker peptide and the plasmid vector recovered by the same enzyme digestion.

The ligation system was: 2 μL of vector, 5 μL of target gene, 1 μL of 10× ligation buffer, 1 μL of T4 DNA ligase, and finally added to 10 μL with ddH ₂ O. Connect at 16°C for 12-16h.

5. Preparation of E. coli competent cells (calcium chloride method)

Dip the Escherichia coli strains frozen at -70°C with a sterile inoculating loop, streak it on an antibiotic-free LB agar plate, and culture at 37°C for about 16 hours. Pick a single colony, inoculate it into 3 mL of LB liquid medium without antibiotics, and incubate overnight at 37°C with shaking. The next day, 3 mL of bacterial solution was added to 100 mL of antibiotic-free LB medium preheated to 37 °C, and vigorously shaken at 37 °C (250 rpm) to culture to an OD600 value of about 0.4 to 0.6 (about 2.5 to 3 h). Transfer the bacterial culture solution to two sterile and ice-cold polypropylene centrifuge tubes under aseptic conditions (all the following operations need to be sterile and performed in an ice-water bath), ice-bath for 10 min, and allow the culture to cool to a 0°C. Centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant, resuspend the pellet with 10 mL of ice-cold CaCl ₂ solution (75 mM CaCl ₂ , 10 mmol/L Tris-Cl pH6.5) solution, and take an ice bath for 10 min. Centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant, and resuspend each tube with 2 mL of ice-cold CaCl ₂ preservation solution (75 mM CaCl ₂ , 10 mmol/L Tris-Cl pH6.5, 15% (v/v) glycerol) solution Precipitate, use sterile pipette tips to distribute competent cells into sterile microcentrifuge tubes, 100 μL per tube, and store them in a -70°C ultra-low temperature refrigerator for later use.

6. Conversion

Take 5 μL of the ligation product obtained in the above step 4 to transform E. coli DH5α competent cells by heat shock method.

The specific method is: add 5 μL of the ligation product to 100 μL of competent cells, ice bath for 30 minutes, 42°C for 90s, ice bath for 3-5 minutes, spread evenly on LB agar plates containing X-gal, IPTG and ampicillin, and culture at 37°C for 12- 16h; inoculation culture: pick a single colony, inoculate it in 5ml of LB (containing 50μg/ml ampicillin) medium, and culture with shaking at 200rpm at 37°C for 12h.

7. Plasmid tipping

The positive plasmids obtained by screening were rapidly identified by PCR method with plasmid DNA rapid preparation kit. Use V-gene company's plasmid mini-pump kit to mini-pump the plasmid. Proceed as follows: collect 1 mL of overnight culture, centrifuge at 13,000 rpm for 30 sec, add 150 μL of solution I, vortex to suspend bacteria, add 150 μL of solution II, invert several times, lyse bacteria at room temperature for 3-5 min, and add 400 μL of solution III, protein was precipitated at room temperature for 5 min, then centrifuged at 13,000 rpm for 10 min, the supernatant was carefully aspirated into DNA-prep tube, centrifuged at 4,000 rpm for 1 min, washed once with 500 μL buffer W1 and twice with 500 μL buffer W2, centrifuged at 13,000 rpm for 1 min, added 40-50 μL of TE was placed at the bottom of the column, placed at room temperature for 1 min, centrifuged at 13,000 rpm for 1 min, and the centrifuged liquid was the plasmid DNA solution.

8. Identification of recombinant plasmids

The obtained recombinant plasmid was sent to Shanghai United Gene Company for sequencing.

2. Results.

1. Conversion results.

The target fragment obtained by ligation was ligated into the pET-28a(+)Fc plasmid, and the plasmid was picked from the white colony and double-enzyme digested. The obtained DNA bands were all as expected, as shown in Figure 1.

2. Recombinant plasmid sequencing results

The obtained recombinant plasmid was sent to Shanghai United Gene Company for sequencing. The results showed that the sequence reading frame and direction were correct, indicating that the target gene had been correctly inserted into the expression plasmid.

Example 2

Expression and purification of recombinant protein in host bacteria

1. Methods.

1. Conversion

The positive recombinant plasmid p was transformed into E. coli competent BL21 (DE3) by heat shock method, and cultured on LB resistant agar plates (containing 50 μg/ml ampicillin) at 37°C for 12-16h. Pick the recombinant plasmid transformed bacteria of a single strain, inoculate 10ml of LBG medium containing 50μg/ml ampicillin, shake and cultivate at 37°C until the OD ₆₀₀ reaches 0.4-0.6, add IPTG to a final concentration of 0.3mmol/L, 200rpm Continue to cultivate for 3.5h.

2. Induced expression

Take 1 ml of the above induction product, centrifuge the induced bacterial culture at 5000 rpm, discard the supernatant, add 100 μL of PBS to the precipitate and mix well, add 100 μL of 2×SDS loading buffer and mix well, boil for 5 minutes, centrifuge at 8000 rpm for 5 minutes at 4°C, and take 20 μL of the supernatant. SDS-PAGE electrophoresis was performed.

3. Fusion protein expression analysis

After the induced bacteria were centrifuged at 6000 rpm for 5 min, discarded the supernatant, suspended in 1/10 V PBS, and sonicated at a power of 300 W, super 5 sec, stopped for 10 sec, and sonicated 50 times in total. After sonication, centrifuge at 12,000 rpm for 10 min, and take the supernatant and pellet for SDS-PAGE respectively.

4. Washing and denaturation of inclusion body recombinant protein

(1) Washing: Resuspend the target protein mainly in the form of inclusion bodies with lysis buffer (50 mmol/L Tris-HCl, pH8.0, 1 mmol/L EDTA, 100 mmol/1 L NaCl), pass through 2% Triton X /100 (polyethylene glycol octyl phenyl ether), 0.2% sodium deoxycholate, and 1 mol/L urea to obtain preliminary purification after washing.

(2) Denaturation: The initially purified inclusion bodies were denatured and dissolved with 8 mmol/L urea (25 mmol/L Tris-HCl, pH 8.0, 20 mmol/L DTT) for 6 h.

5. Purify the target protein by SP-sepharose cation chromatography column

(1) The SP-sepharose cation column (1 ml) was connected to the FPLC, and the column was washed with 10 ml of distilled water. The purpose is to flush the 20% ethanol from the column.

(2) Equilibrate the column with 5-10 column volumes of starting buffer solution (25 mmol/L Tris-HCl, pH 8.0, 0.2% dimercaptoethanol, 8 mol/L urea). The flow rate was 1 ml/min.

(3) Load the sample with a syringe or peristaltic pump, and the flow rate is 1 ml/min. Collection across peaks.

(4) Wash with 5-10 column volumes of starting buffer until the absorption at 280nm reaches the baseline level.

(5) Use 10-25 column volumes of elution buffer (25 mmol/L Tris-HCl, pH 8.0, 8 mol/L urea, 1 mol/L NaCl) for linear gradient elution, and collect each elution peak in separate tubes.

(6) The elution buffer is eluted for another 10 column volumes.

(7) 12% SDS-PAGE electrophoresis to determine the components of the target protein.

(8) Dialysis: The components containing the target protein were dialyzed against the starting buffer for 48 hours, and the dialysate was replaced every 8 hours.

The eluted samples were detected by SDS-PAGE, stained with Coomassie brilliant blue, and photographed after destaining with the destaining solution.

6. Determination of purified target protein content

Using BCA protein quantification kit (manufacturer: Pierce, model: 23225), the content of purified fusion protein was determined according to the instructions.

7. Refolding of recombinant protein

Dilution renaturation under different oxidation conditions

(1) Redox agent:

In the purified protein buffer, DTT was added to the final concentration of 10 mmol/L for 1 h, and reduced: oxidized glutathione (2 mmol/L: 0.2 mmol/L) was added for 1 h. According to the results of protein quantification, dilute it with 4mol/L urea to 0.5mg/ml, and gradually decrease the concentration (4mol/L-2mol/L-0mol/L) of urea solution (containing the same concentration of reduced oxidized glutathione) Dialysis renaturation.

(2) Air oxidative renaturation under alkaline conditions:

The purified P-5m-Fc fusion denatured protein was dialyzed into an alkaline 8mol/L urea buffer (25mmol/LTris-HCl, pH10.0), and DTT was added to a final concentration of 10mmol/L for 1 hour. Stir in the air overnight, according to the protein quantification results, first dilute to 0.5mg/ml, and gradually reduce the concentration (4mol/L-2mol/L-0mol/L) of urea buffer solution (25mmol/L Tris-HCl, pH7.2 ) dialysis renaturation.

2. Results.

1. Expression of P-5m-Fc fusion protein

Take the correctly sequenced recombinant plasmid, transform it into E.coli BL21 (DE3), pick a single colony, activate it, and then shake it at 37°C until the OD600 is about 0.6-0.8. IPTG was added to 0.5 mmol/L, and after inducing expression for 4 hours, the bacteria were harvested, the bacteria were disrupted by ultrasonic, and centrifuged to obtain the supernatant and precipitate. The whole cell, supernatant and precipitate were taken for SDS-PAGE. The results are shown in Figure 2, Figure 3, and Figure 4, indicating that the P-5m-Fc fusion protein was mainly expressed in the form of inclusion bodies with a molecular weight of about 34kD.

2. Washing and solubilization of recombinant protein inclusion bodies

Inclusion bodies are mainly composed of recombinant proteins and some bacterial impurities, including RNA polymerase, bacterial membrane proteins, rRNA and plasmid DNA.

On the one hand, the formation of inclusion bodies is conducive to the separation and purification of recombinant proteins and the stability of proteins, but because inclusion bodies are misfolded forms of proteins, they are often inactive and must be denatured and dissolved, renatured under appropriate conditions, and refolded into a correct form. Conformation of an active protein molecule. Generally, inclusion bodies should be washed with low concentrations of denaturant and detergent before solubilization to remove impurities on the surface of inclusion bodies. Washing conditions are best to remove as much impurities as possible without dissolving inclusion bodies. After groping with different washing conditions and concentrations, a series of washing conditions of 2% Triton X-100, 0.2% sodium deoxycholate, and 1 mol/L urea were finally determined, and the inclusion bodies were initially purified, as shown in Figure 5.

3. Purification by SP-sepharose cation column chromatography

The denatured fusion protein was further purified by cationic column chromatography. At pH 8.0, the fusion protein was bound to the cationic resin and eluted with a buffer containing NaCl, as shown in Figure 6.

Example 3

Activity detection of P-5m-Fc fusion protein

1. Western-blotting test

The protein bands (Fc and P-5m-Fc fusion protein) analyzed by SDS-PAGE were transferred to PVDF membrane, blocked with 1% BSA at 4°C overnight, and washed 3 times with 0.05% PBST for 10 min each time. , add 2000 ^× diluted positive serum, act at 37°C for 2 h, wash 3 times with 0.05% PBST, 10 min each time, add 25000 ^× diluted rabbit anti-human IgG enzyme-labeled antibody, wash 3 times with 0.05% PBST, 10 min each time , PIERCE luminescent substrate was added for 3 min at room temperature, exposed in a dark room, and fixed after developing. The results are shown in Figure 6.

The above results indicate that Fc cannot bind to P-5m polyclonal antibody, while P-5m-Fc can specifically bind to P-5m polyclonal antibody.

2. Indirect ELISA test

1. Preparation of polyclonal antibodies against P-5m octapeptide

(1) Antigen preparation

Antigen-prepared polypeptides were synthesized by 9-fluoromethoxycarbonyl (Fmoc) solid-phase synthesis method to synthesize polypeptide fragments, purified by high performance liquid phase method, and the purified samples were analyzed by HPLC, and the purity was >97%. The polypeptide is linked to the carrier protein-hemocyanin.

(2) Animal immunity

250 μl of the purified polypeptide antigen solution was mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and the New Zealand white rabbits were immunized by back multi-point injection. After 2 weeks, the peptide antigen solution was taken and an equal volume of incomplete Freund's adjuvant was used to boost the immunization. After that, the immunization was once every two weeks. The whole process was immunized 5 times. Blood was tested in the ear vein 5-7 days after the last immunization. Indirect ELISA method After the serum was detected to reach the ideal titer, the carotid artery was bled, and the rabbit serum was collected.

(3) Collection and purification of immunized rabbit serum

Rabbit serum was collected by the common carotid artery bleed method.

Preliminary purification of rabbit serum by salting out method:

A. Mix equal volumes of 10 mL serum and normal saline, add saturated ammonium sulfate dropwise while stirring to make the final concentration 50%, overnight at 4°C to fully precipitate the protein.

B. Centrifuge at 3000rpm/min for 20min, and discard the supernatant. Dissolve the precipitate in 10 mL of physiological saline, add saturated ammonium sulfate dropwise with stirring to make the final concentration 33%, and precipitate at 4°C for more than 3 hours.

C. Centrifuge at 3000rpm/min for 20min, discard the supernatant.

D. The obtained precipitate was dissolved to 5 mL with PBS, put into a dialysis bag, and dialyzed at 4° C. with PBS 20 times larger than its volume for 1-2 days, during which the PBS was replaced several times until the outer dialysate liquid was detected by Naphthalene reagent without yellow.

E. The preliminarily purified rabbit serum was separated and purified by C18 reversed-phase chromatography column, and then freeze-dried. (Quantitative lyophilized powder of affinity purified antibody IgG, 1 mg/vial, stored at -20°C).

(4) Determination of serum titer by indirect ELISA

Before the start of the injection immunization procedure and 1 week after each injection immunization, 1 mL of blood was collected from the ear vein of the rabbit, and the supernatant was centrifuged to measure the serum titer.

A. Coating: Dissolve the synthesized polypeptide in 0.1 mol/L carbonate buffer (pH 9.6) to prepare 100 μg/ml antigen coating solution, coat 100 μl ELISA plate, and set up a negative control (1 : 200 dilution of normal rabbit serum) and blank control (without primary antibody), each with 3 replicate wells, 100 μL/well, coated at 37°C for 3 hours;

B. Washing: 250μL/well washing solution, wash 3 times, 5min each time;

C. Blocking: 200μL/well blocking solution, 37℃ for 2h;

D. Washing: 250μL/well washing solution, wash 3 times, 5min each time;

E. Add primary antibody: add 1:200 diluted rabbit immune serum, 100 μL/well, and incubate at 37°C for 1 h;

F. Washing: 250μL/well washing solution, wash 3 times, 5min each time;

G. Add secondary antibody: add 1:10 000 goat anti-mouse IgG-HRP, 100 μL/well, incubate at 37°C for 1 h;

H. Washing: 250μL/well washing solution, wash 5 times, 5min each time;

I. Color development: add OPD working solution, 100 μL/well, and protect from light for 10-20 minutes;

J. Termination reaction: 2 mol/L sulfuric acid, 50 μL/well.

The A492 value was determined by the microplate reader, and the value of (tested sample value-blank value)/(negative control value-blank value), that is, P/N≥2.1, was positive.

2. Preparation and titer of P-5m octapeptide antibody

The results showed that the P-5m peptide had good immunogenicity. The antibody titer in the body began to rise 2 weeks after the first immunization, that is, the 3rd week, and the 9th week after the five active immunizations ended. Polypeptide antigen showed strong positive reaction, and the rabbit serum of control group did not have immune reaction with peptide. The serum obtained after immunization of rabbits was double-diluted. With the increase of serum dilution, the OD value of the reaction well decreased. P/N≧2.1 was positive. High titer polyclonal antibody.

3. Detection

The purified P-5m-Fc fusion protein was coated with 10 μg/mL microtiter plate, coated overnight at 4 °C, and then washed three times with 0.05% PBST for 3 min each time, and added 1% BSA at 37 °C. Blocked for 2h, washed 3 times with 0.05% PBST for 3 min each, added 50× diluted negative and positive serum, acted at 37°C for 1 h, washed 3 times with 0.05% PBST for 3 min each, added 10000 ^× diluted rabbit anti-human IgG enzyme-labeled antibody, reacted at 37°C for 0.5h, washed 3 times with 0.05% PBST for 3 minutes each time, added substrate OPD, reacted at 37°C for 15min, terminated with 2mol/LH ₂ SO ₄ , and read the OD490 value with an ELx800 microplate reader .

The results showed that a high titer of P-5m polyclonal antibody was obtained.

3. Functional study of P-5m-Fc peptibody against tumor metastasis

1. Expression and purification of Fc control protein

In order to confirm that the peptide part is functioning and the control Fc protein is expressed at the same time, the constructed pET-28a-Fc fusion expression plasmid was transformed into BL21 (DE3), and the expression was induced by IPTG (Figure 2, 3), the cells were collected, and ultrasonically disrupted. Bacteria, centrifuged to collect the inclusion body part, washed and denatured and dissolved in urea solution as the fusion protein, and purified.

2. Inhibitory effect of fusion protein on matrix membrane invasion of HCCLM3 cells

(1) Method

A. Coat Matrigel-free Transell chambers with basement membrane: Dilute Matrigel to 200 μg/ml with serum-free RPMI 1640 medium on ice, add 100 μl of mixed liquid to each upper chamber, and place in a CO ₂ constant temperature incubator After 30min of coagulation, suck off the excess liquid.

B. Hydration of basement membrane: 500 μl of serum-free RPMI 1640 medium was added to the upper and lower chambers, respectively, and placed in a CO ₂ constant temperature incubator for hydration for 30 min.

C. Preparation of cell suspension: Digest HCCLM3 cells in logarithmic growth phase, discard the original culture medium, dilute the cells to 5×10 ⁵ cells/ml with serum-free medium containing 10 mg/ml BSA, and add Final concentrations of P-5m were 0 μM, 10 μM and 100 μM.

D. Inoculating cells: Inoculate 200 μl of cell suspensions of different groups into the upper chamber of the transwell, add 500 μl of RPMI 1640 medium containing 10% FBS to the lower chamber, and culture in a CO ₂ constant temperature incubator for 24 h.

E. Fixation: Aspirate the upper chamber liquid, gently wipe off the cells and Matrigel glue on the membrane with a cotton swab, fix the cells under the membrane with ice-cold methanol for 10 minutes, and air dry.

F. Staining: stain with 0.1% crystal violet for 15 minutes at room temperature, wash with sterile water 3 times, and air dry.

G. Take pictures under an inverted microscope (400×), count the number of cells in 5 fields of view in each well, and compare the differences in the invasiveness of cells in each group. The experiment was repeated three times.

(2) Results

The above-mentioned transwell invasion assay was used to investigate the inhibitory effect of P-5m-Fc fusion protein on the migration of hepatoma cells after 48 hours of treatment.

As shown in Figure 7-8, the results of the invasion assay of HCCLM3 cells showed that the number of penetrating cells in the 10 μM P-5m-Fc treatment group was 31.6% less than that in the control group, and the number of penetrating cells in the 100 μM P-5m treatment group was 43.4% less than that in the control group. %, the difference was statistically significant (P≤0.05); the number of transmembrane cells was 11.9% less than that of the control group after co-treatment with 100 μM P-5m-Fc and P-5m polyclonal antibody, and the difference was not statistically significant. The invasion experiment further clarified the effect of P-5m on tumor cell invasion after antibody blocking, and the results showed that the antibody could block the inhibitory ability of P-5m-Fc on tumor cell invasion.

Moreover, it can also be seen from the experimental results that the number of penetrating cells in the 10 μM P-5m-Fc treatment group was 13.0% less than that in the same dose of P-5m treatment group, and the difference was statistically significant (P≤0.05). ); the number of penetrating cells in the 100 μM P-5m-Fc treatment group was 12.3% less than that in the same dose of P-5m treatment group, and the difference was statistically significant (P≤0.05).

It can be seen from the above results that the expressed and purified P-5m-Fc fusion protein has better anti-tumor cell invasion activity, and its anti-tumor cell invasion effect is better than that of P-5m octapeptide.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (5)

1. A P-5m-Fc fusion protein, characterized in that it is formed by connecting a functional unit to the Fc portion of human immunoglobulin IgG1, wherein the functional unit is composed of an amino acid sequence shown in SEQ ID NO.1 and a linker The linker is composed of six consecutively connected glycines, the P-5m-Fc fusion protein has at least one of the functional units, and when the P-5m-Fc fusion protein has multiple When there are several functional units, a plurality of functional units are connected in sequence and then connected to the Fc part of the immunoglobulin; The P-5m-Fc fusion protein is prepared by the following method: (1) construct the expression gene, and insert the above-mentioned expression gene into the carrier, construct the recombinant expression vector; The sequence of the expressed gene is: composed of the nucleotide sequence of the functional unit of the nucleotide sequence shown in SEQ ID NO. 2 and the nucleotide sequence of the Fc part of the encoding immunoglobulin; (2) transferring the above-mentioned recombinant expression vector into a host cell, and obtaining a positive expression vector through screening; (3) transferring the above-mentioned positive expression vector into expression cells, and inducing expression of the expressed gene sequence in step (1); (4) Purification: collect the induced-expressed cells, suspend them in phosphate buffer, sonicate and centrifuge to obtain the P-5m-Fc fusion protein expressed by inclusion bodies; resuspend the above inclusion bodies in lysis buffer, The lysis buffer includes 50 mmol/L pH8.0 Tris-HCl, 1 mmol/L EDTA, 100 mmol/1 L NaCl, and then passes through 2% Triton X/100 by volume percentage, and the mass percentage concentration is 0.2% The sodium deoxycholate was washed with 1mol/L urea for preliminary purification; The preliminarily purified inclusion bodies were denatured and dissolved with 25 mmol/L Tris-HCl buffer containing 8 mmol/L urea and 20 mmol/L DTT at pH 8.0; Then, the above-mentioned P-5m-Fc fusion protein is purified by SP-sepharose cation chromatography column, and then renatured by redox agent or air oxidation under alkaline conditions; The conditions for renaturation of the redox agent are: in the purified P-5m-Fc fusion protein buffer, add DTT to a final concentration of 10 mmol/L, and then add reduced glutathione and oxidized glutathione To the concentration of 2mmol/L and 0.2mmol/L, respectively, the P-5m-Fc fusion protein was diluted to 0.5mg/ml with 4mol/L urea, and the concentration was changed from 4mol/L, 2mol/L, 0mol/L urea to 0.5mg/ml. Dialysis and renaturation of buffer solution, the urea solution contains the same concentration of reduced and oxidized glutathione; The conditions of air oxidative renaturation under the described alkaline conditions are as follows: the purified P-5m-Fc fusion protein is dialyzed into a 25mmol/L Tris-HCl buffer of pH10.0 with a urea content of 8mol/L, and DTT is added. To the final concentration of 10 mmol/L, exposed to the air and stirred overnight, the P-5m-Fc fusion protein was diluted to 0.5 mg/ml, and renatured by dialysis with urea buffer solution with a concentration of 4 mol/L, 2 mol/L and 0 mol/L. ; An active P-5m-Fc fusion protein with the correct conformation was obtained by renaturation. 2. a preparation method of the described P-5m-Fc fusion protein of claim 1, is characterized in that, comprises the following steps: (1) Construction of the expression gene according to claim 1, the sequence of the expression gene is: the nucleotide sequence of the functional unit of the nucleotide sequence shown in SEQ ID NO. 2 and the Fc encoding immunoglobulin Part of the nucleotide sequence combination is formed; and the above-mentioned expression gene is inserted into the vector to construct a recombinant expression vector (2) transferring the above-mentioned recombinant expression vector into a host cell, and obtaining a positive expression vector through screening; (3) transferring the above-mentioned positive expression vector into expression cells, and inducing expression of the expressed gene sequence in step (1); (4) Purification: collect the induced-expressed cells, suspend them in phosphate buffer, sonicate and centrifuge to obtain the P-5m-Fc fusion protein expressed by inclusion bodies; resuspend the above inclusion bodies in lysis buffer, The lysis buffer includes 50 mmol/L pH8.0 Tris-HCl, 1 mmol/L EDTA, 100 mmol/1 L NaCl, and then passes through 2% Triton X/100 by volume percentage, and the mass percentage concentration is 0.2% The sodium deoxycholate was washed with 1mol/L urea for preliminary purification; The preliminarily purified inclusion bodies were denatured and dissolved with 25 mmol/L Tris-HCl buffer containing 8 mmol/L urea and 20 mmol/L DTT at pH 8.0; Then, the above-mentioned P-5m-Fc fusion protein is purified by SP-sepharose cation chromatography column, and then renatured by redox agent or air oxidation under alkaline conditions; The conditions for renaturation of the redox agent are: in the purified P-5m-Fc fusion protein buffer, add DTT to a final concentration of 10 mmol/L, and then add reduced glutathione and oxidized glutathione To the concentration of 2mmol/L and 0.2mmol/L, respectively, the P-5m-Fc fusion protein was diluted to 0.5mg/ml with 4mol/L urea, and the concentration was changed from 4mol/L, 2mol/L, 0mol/L urea to 0.5mg/ml. Dialysis and renaturation of buffer solution, the urea solution contains the same concentration of reduced and oxidized glutathione; The conditions of air oxidative renaturation under the described alkaline conditions are as follows: the purified P-5m-Fc fusion protein is dialyzed into a 25mmol/L Tris-HCl buffer of pH10.0 with a urea content of 8mol/L, and DTT is added. To the final concentration of 10 mmol/L, exposed to the air and stirred overnight, the P-5m-Fc fusion protein was diluted to 0.5 mg/ml, and renatured by dialysis with urea buffer solution with a concentration of 4 mol/L, 2 mol/L and 0 mol/L. ; An active P-5m-Fc fusion protein with the correct conformation was obtained by renaturation. 3. the preparation method of P-5m-Fc fusion protein according to claim 2, is characterized in that, In step (1), construct the recombinant expression vector by the following method: first obtain the gene fragment that the base sequence is formed into SEQ ID NO.2, with BamH I and Nde I double enzyme cut pET-28a (+) Fc plasmid, reclaim The enzyme-cut vector fragment, the gene fragment of the above-mentioned SEQ ID NO. 2 is inserted into the above-mentioned vector by DNA ligase to obtain a recombinant plasmid, which is a recombinant expression vector; In step (2), the positive expression vector is obtained by the following method: the ligation product is transferred into Escherichia coli competent DH5α by heat shock method, and coated on X-gal, isopropyl-β-D-thiohalogen On the LB agar medium of lactoside and ampicillin, the positive plasmid is obtained by screening, which is the positive expression vector; In step (3), the specific method for inducing expression is: transfer the positive plasmid obtained in step (2) into E. coli E.coli BL21 (DE3) competent cells by heat shock method, and coat it on ampicillin-containing cells. On the LB agar medium of penicillin, culture; pick a single colony and inoculate it into LBG medium containing ampicillin, culture, add isopropyl-β-D-thiogalactoside after culture, and then induce culture; obtain P- 5m-Fc fusion protein. 4. the preparation method of P-5m-Fc fusion protein according to claim 3, is characterized in that, In step (2), the ligation product is transferred into Escherichia coli competent DH5α by heat shock method, and coated on LB agar medium containing X-gal, isopropyl-β-D-thiogalactoside and ampicillin After culturing for 12-16 hours, pick a single colony, inoculate it in LB medium containing 50 μg/ml ampicillin, and screen to obtain a positive plasmid; In step (3), the positive plasmid obtained in step (2) was transferred into Escherichia coli E.coliBL21 (DE3) competent cells by heat shock method, and spread on the LB agar medium containing 50 μg/ml ampicillin , cultivate for 12-16h; pick a single colony and inoculate it into LBG medium containing 50μg/ml ampicillin, shake and cultivate until the OD ₆₀₀ reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside to The final concentration is 0.2-0.6 mmol/L, induce culture, centrifuge the culture, take the precipitate, add phosphate buffer and loading buffer, boil and centrifuge again to obtain the P-5m-Fc fusion protein. 5. The application of the P-5m-Fc fusion protein of claim 1 in the preparation of a medicine for inhibiting the metastasis of liver cancer cells.

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