CN106046177B

CN106046177B - P-5m-Fc fusion protein and expression gene, preparation method and application thereof

Info

Publication number: CN106046177B
Application number: CN201610695327.4A
Authority: CN
Inventors: 韩笑; 杜培革; 安丽萍; 徐广宇; 孙静波; 苑广信; 郭笑; 赵南晰; 王曼力; 李洪宇; 盛瑜
Original assignee: Beihua University
Current assignee: Beihua University
Priority date: 2016-08-19
Filing date: 2016-08-19
Publication date: 2020-02-21
Anticipated expiration: 2036-08-19
Also published as: CN106046177A

Abstract

The invention relates to a P-5m-Fc fusion protein, an expression gene, a preparation method and application thereof, belonging to the technical field of medical biology. The P-5m-Fc fusion protein is formed by connecting a functional unit and an Fc part of human immunoglobulin IgG1, wherein the functional unit is formed by connecting an amino acid sequence shown in SEQ ID NO.1 and a linker, the linker is a polypeptide formed by six sequentially connected glycines, at least one functional unit is arranged in the P-5m-Fc fusion protein, and when a plurality of functional units are arranged in the P-5m-Fc fusion protein, the functional units are sequentially connected and then connected with the Fc part of the immunoglobulin. The P-5m-Fc fusion protein fuses and expresses P-5m and Fc fragment of human IgG1 to prepare a peptide body, which not only can improve the drug effect of P-5m octapeptide, but also can obviously prolong the half-life period of the drug and obtain good drug property. And experimental research proves that the compound has a remarkable function of resisting tumor metastasis.

Description

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

Technical Field

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

Background

Current studies indicate that tumor metastasis is the underlying cause of loss of life in tumor patients. The P-5m octapeptide is a polypeptide with eight amino acids derived from the fifth domain (D5) of kininogen macromolecule, which is the shortest polypeptide which is found to be derived from D5 and has the effect of effectively resisting tumor metastasis.

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

However, the P-5m octapeptide has the defects of poor stability and short half-life after being administered, and is still difficult to apply in clinic at present.

Disclosure of Invention

In view of the above, there is a need to provide a P-5m-Fc fusion protein, which can prolong the stability and half-life of P-5m octapeptide and enhance the pharmaceutical properties thereof.

A P-5m-Fc fusion protein is formed by connecting a functional unit and an Fc part of human immunoglobulin IgG1, wherein the functional unit is formed by connecting an amino acid sequence shown in SEQ ID NO.1 and a linker, the linker is a polypeptide formed by six sequentially connected glycines, at least one functional unit is arranged in the P-5m-Fc fusion protein, and when a plurality of functional units are arranged in the P-5m-Fc fusion protein, the functional units are sequentially connected and then connected with the Fc part of the immunoglobulin.

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

The invention also discloses an expression gene for coding the P-5m-Fc fusion protein, and the sequence of the expression gene is as follows:

A) is composed of a combination of a nucleotide sequence of a functional unit and an Fc part nucleotide sequence of a coded immunoglobulin; or

B) A sequence which 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 obtained by modifying the nucleotide sequence shown in the A) or the B) by substitution, deletion and addition of one or more bases.

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

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

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

The invention also discloses a preparation method of the P-5m-Fc fusion protein, which comprises 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 recombinant expression vector into a host cell, and screening to obtain a positive expression vector;

(3) transferring the positive expression vector into an expression cell, and performing induced expression on the expression gene sequence in the step (1) to obtain the gene.

In one embodiment, in step (1), the recombinant expression vector is constructed by: firstly, obtaining a gene fragment with a base sequence component of SEQ ID NO.2, carrying out double digestion on pET-28a (+) Fc plasmid by using BamH I and Nde I, recovering a digested vector fragment, and inserting the gene fragment of SEQ ID NO.2 into the vector through DNA ligase to obtain a recombinant plasmid, namely a recombinant expression vector;

transferring the ligation product into escherichia coli competence DH5 α by a heat shock method, coating the escherichia coli competence DH5 α on an LB agar culture medium containing X-gal, isopropyl- β -D-thiogalactoside (IPTG) and ampicillin, and screening to obtain a positive plasmid, namely the positive expression vector;

in the step (3), the specific method for inducing expression is that the positive plasmid obtained in the step (2) is transferred into escherichia coli E.coli BL21(DE3) competent cells through a heat shock method, the competent cells are coated on an LB agar culture medium containing ampicillin for culture, a single colony is selected and inoculated into an LBG culture medium containing ampicillin for culture, isopropyl- β -D-thiogalactoside is added after culture, and then induction culture is carried out, so as to obtain the P-5m-Fc fusion protein.

In one embodiment, in step (2), the ligation product is transferred into escherichia coli competent DH5 α by a heat shock method, spread on LB agar medium containing X-gal, isopropyl- β -D-thiogalactoside and ampicillin, cultured for 12-16h, then single colony is picked up and inoculated on LB medium containing 50 mug/ml ampicillin, and positive plasmid is obtained by screening;

in the step (3), the positive plasmid obtained in the step (2) is transferred into escherichia coli E.coli BL21(DE3) competent cells through a heat shock method, and the competent cells are coated on an LB agar culture medium containing 50 mu g/ml ampicillin and cultured for 12-16 h; single colonies were picked, inoculated into LBG medium containing 50. mu.g/ml ampicillin, and shake-cultured to OD₆₀₀Adding isopropyl- β -D-thiogalactoside to a final concentration of 0.2-0.6mmol/L when the concentration reaches 0.4-0.6, performing induction culture, centrifuging the culture, taking the precipitate, adding phosphate buffer solution and loading buffer solution, boiling, and centrifuging again to obtain the P-5m-Fc fusion protein.

In one embodiment, the method further comprises the step (5) of purifying: collecting thalli after induction expression, suspending the thalli by using a phosphate buffer solution, carrying out ultrasonic crushing and centrifuging to obtain P-5m-Fc fusion protein expressed by inclusion bodies; the inclusion bodies are resuspended by lysis buffer solution, then washed by Triton X/100 (polyethylene glycol octyl phenyl ether), sodium deoxycholate and urea for primary purification, the inclusion bodies after primary purification are denatured and dissolved by Tris-HCl buffer solution containing urea and DTT (dithiothreitol), and then the active P-5m-Fc fusion protein with correct conformation is obtained by redox renaturation or air redox renaturation under alkaline condition.

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

In one embodiment, the step (5) of purifying comprises: resuspending the inclusion body by a lysis buffer solution, wherein the lysis buffer solution comprises 50mmol/L Tris-HCl with pH8.0, L mmol/L EDTA, 100mmol/1L NaCl, and then washing by 2% volume percentage of Triton X/100, 0.2% mass percentage of sodium deoxycholate and 1mol/L urea for preliminary purification;

performing denaturation and dissolution on the primarily purified inclusion body by using 25mmol/L Tris-HCl buffer solution with pH8.0, 8mmol/L urea and 20mmol/L DTT;

purifying the P-5m-Fc fusion protein by using an SP-sepharose cation chromatographic column, and then carrying out redox renaturation or air oxidation renaturation under the alkaline condition;

the condition of the redox agent renaturation is as follows: adding DTT into the purified P-5m-Fc fusion protein buffer solution to a final concentration of 10mmol/L, then adding reduced glutathione and oxidized glutathione to concentrations of 2mmol/L and 0.2mmol/L respectively, diluting the P-5m-Fc fusion protein to 0.5mg/ml by using 4mol/L urea, and dialyzing for renaturation by using a urea buffer solution with the concentration gradually reduced from 4mol/L to 0mol/L, wherein the urea solution contains reduced glutathione and oxidized glutathione with the same concentration;

the air oxidation renaturation condition under the alkaline condition is as follows: dialyzing the purified P-5m-Fc fusion protein into 25mmol/L Tris-HCl buffer solution with 8mol/L urea content and pH value of 10.0, adding DTT to the final concentration of 10mmol I/L, exposing the solution to the air, stirring the solution overnight, diluting the P-5m-Fc fusion protein to 0.5mg/ml, and dialyzing and renaturing the solution by urea buffer solution with the concentration gradually reduced from 4mol/L to 0 mol/L;

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

The refolding protein has better renaturation effect, and the purity of the fusion protein can be further improved by purifying the refolding protein by using an SP-sepharose cation chromatographic column.

The invention also discloses application of the P-5m-Fc fusion protein in preparing a medicament for inhibiting liver cancer cell metastasis.

Particularly has better inhibition effect on HCCLM3 cell invasion plasma membrane, and can be used for inhibiting the transfer of human liver cancer HCCLM3 cells.

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

according to the P-5m-Fc fusion protein, the P-5m and the Fc fragment of human IgG1 are fused and expressed to prepare the peptide body, so that the drug effect of the P-5m octapeptide can be improved, the half-life period of the drug can be remarkably prolonged, and good drug-forming property can be obtained. And experimental research proves that the compound has a remarkable function of resisting tumor metastasis.

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

Furthermore, the optimal conditions for refolding a misfolded protein present in an inclusion body into an active P-5m-Fc fusion protein having a correct conformation were obtained by optimizing and screening the renaturation conditions of the P-5m-Fc fusion protein expressed in the inclusion body.

Drawings

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

In the figure: 1 is pET-28a (+) P-5m-Fc plasmid; 2 is the enzyme cutting result; m is DL2000DNA Marker.

FIG. 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 under the condition of non-induction in Escherichia coli, 3 and 4 are the expression of pET-28a (+) P-5M-Fc under the condition of non-induction in Escherichia coli, M is Protein Marker, and the arrow indicates P-5M-Fc fusion Protein electrophoresis band.

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

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

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

In the figure: 1 and 2 are pET-28a (+) P-5m-Fc expression proteins in supernatant after bacterial liquid cracking; 3 is pET-28a (+) expression protein in the bacteria liquid cracking sediment; m is Protein Marker, and an arrow indicates a P-5M-Fc fusion Protein electrophoresis band.

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

In the figure: 1.2 and 3 are pET-28a (+) P-5m-Fc expression proteins in supernatant after bacterial liquid cracking; 4 and 5 are pET-28a (+) P-5m-Fc protein after primary purification; m is Protein Marker, and an arrow indicates a P-5M-Fc fusion Protein electrophoresis band.

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

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

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

In the figure: control represents blank Control; p-5m-Fc 10. mu. mol indicates the result after treatment with P-5m-Fc at a final concentration of 10. mu. mol; p-5m-Fc 100. mu. mol indicates the result after treatment with P-5m-Fc at a final concentration of 100. mu. mol; p-5m-Fc 100. mu. mol + Ab indicates the result after co-treatment with P-5m-Fc and P-5m polyclonal antibody at a final concentration of 100. mu. mol; p-5m 10. mu. mol shows the result after treatment with P-5m at a final concentration of 10. mu. mol; p-5m 100. mu. mol shows the result after treatment with P-5m at a final concentration of 100. mu. mol.

FIG. 8 is a comparison of the ability of P-5m-Fc to inhibit the invasion of HCCLM3 cells through a transwell chamber in example 3.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

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

Firstly, a method is provided.

1. Extraction and purification of bacterial plasmids

After analysis, the amino acid sequence of P-5m is GHGKHKNK, the inventor synthesizes positive and negative chains of P-5m peptide gene by using codon preferred by Escherichia coli after a large amount of experimental screening, ATG is added at the 5 'end, 6 glycine codons are added at the 3' end (hereinafter, GGT GGT GGT GGT GGT GGT part), and in order to increase the titer, the P-5m peptide gene and the glycine codons are repeated for two cycles, and Nde I recognition site (CAT ATG) and BamH I recognition site (GGA TCC) are respectively generated at the 5 'end and the 3' end after annealing: plus 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' (i.e., the nucleotide sequence set forth in SEQ ID NO. 2); 5'-CGC GGA TCC CTT GTT CTT ATC TTT GCC ATC GCC CAT ACC ACC ACC ACCACC ACC CTT GTT CTT ATC TTT GCC ATC GCC CAT ATG G-3' are provided.

Subsequently, the digestion was carried out according to the instructions provided by the Nde I and BamH I restriction enzyme kits (manufacturer: Promaga, model: R4024 and R6801) to double-digest pET-28a (+) Fc plasmid (source: prepared by reference to the following: fusion expression of TPO mimetic peptide and human IgG1Fc fragment and biological characteristics thereof: Biotechnology, 2002,18(4):424 and 430) with Nde I and BamH I, wherein the plasmid containing the full-length Fc fragment, pCDNA3.1-hIgG 1Fc was purchased from Beijing Pompe (Cat.) K.VIY 0062hIgG1 Fc).

In the enzyme digestion step, single enzyme digestion is firstly carried out, then DNA is extracted, precipitated and dissolved in TE, and then another enzyme digestion is carried out. The enzyme digestion reaction is carried out according to the following system: PCR was performed to recover 10. mu.L of product, 10 XH buffer 2. mu.L/10 XT buffer 3. mu.L, two endonucleases, each L. mu.L, and ddH was finally added₂Adding O to 20 μ L, performing enzyme digestion in water bath at 37 deg.C for 1.5h, and inactivating at 65 deg.C for 15 min. And (5) carrying out 0.8% agarose gel electrophoresis identification.

2. Glass milk method for rapid in-gel recovery of DNA

And (3) carrying out agarose gel electrophoresis on the plasmid double digestion product, wherein the agarose gel electrophoresis of 1.2% is used for recovering the annealing fragment containing the P-5m and the connexon, and the agarose gel electrophoresis of 0.8% is used for recovering the digested vector fragment.

3. Annealing of DNA fragments

Synthesizing the positive and negative chains of the P-5m peptide gene by using codons preferred by escherichia coli, dissolving the positive and negative chains into 0.1 pmol/. mu.L of sterile water, mixing the positive and negative chain oligonucleotides with the same amount, adding an annealing buffer solution to 1x (10mmol/LTris, pH7.5-8.0, 50mmol/L NaCl, 1mmol/L EDTA), thermally denaturing at 95 ℃ for 5min, and slowly cooling to room temperature.

4. DNA sticky end ligation

The gene double strand of the P-5m and the linker peptide is connected by T4DNA ligase, and the recovered plasmid vector is digested by the same enzyme.

The connecting system is as follows: 2. mu.L vector, 5. mu.L target gene, 10 Xligation buffer 1. mu.L T4DNA ligase 1. mu.L, and ddH₂O was added to 10. mu.L. The ligation was carried out at 16 ℃ for 12-16 h.

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

The strain of Escherichia coli frozen at-70 deg.C is dipped in an aseptic inoculating loop, streaked and inoculated on LB agar plate without antibiotic, and cultured at 37 deg.C for about 16 h. A single colony was picked and inoculated into 3mL of LB liquid medium containing no antibioticsShaking and culturing overnight at 37 ℃. The next day, 3mL of the cell suspension was added to 100mL of LB medium without antibiotics preheated to 37 ℃ and cultured with vigorous shaking (250rpm) at 37 ℃ until the OD600 value was about 0.4-0.6 (about 2.5-3 hours). The bacterial cultures were aseptically transferred to two sterile and ice-precooled polypropylene centrifuge tubes (both aseptically and in an ice-water bath) and ice-cooled for 10min to cool the cultures to 0 ℃. Centrifuging at 4 deg.C and 5000rpm for 10min, discarding supernatant, and pre-cooling with 10mL of ice-cooled CaCl₂Solution (75mM CaCl)₂10mmol/L Tris-Cl pH6.5) solution, resuspending the pellet in ice bath for 10 min. Centrifuging at 4 deg.C and 5000rpm for 10min, discarding supernatant, and adding 2mL of CaCl pre-cooled with ice₂Preservation solution (75mM CaCl)₂10mmol/L Tris-Cl pH6.5, 15% (v/v) glycerol) solution, resuspending the pellet per tube, subpackaging the competent cells in sterile microfuge tubes with sterile pipette tips, 100 μ L per tube, and freezing at-70 deg.C in an ultra-low temperature refrigerator for later use.

6. Transformation of

mu.L of the ligation product obtained in step 4 above was used to transform E.coli DH5 α competent cells by heat shock.

The specific method comprises the following steps: adding 5 μ L of the ligation product into 100 μ L of competent cells, ice-bathing for 30min at 42 ℃ for 90s, ice-bathing for 3-5min, uniformly coating on LB agar plate containing X-gal, IPTG and ampicillin, and culturing at 37 ℃ for 12-16 h; inoculating culture, picking single colony, inoculating in 5ml LB (containing 50. mu.g/ml ampicillin) culture medium, and shaking culturing at 37 deg.C 200rpm for 12 h.

7. Plasmid small grape

The positive plasmid obtained by rapid identification and screening by a PCR method is extracted by using a small rapid preparation kit of plasmid DNA. The plasmid was minified using a V-gene plasmid miniprep kit. The method comprises the following steps: collecting 1mL of overnight culture, centrifuging at 13000rpm for 30sec, adding 150 mu L of solution I, vortexing and shaking the suspended bacteria, adding 150 mu L of solution II, inverting the solution several times, lysing the bacteria at room temperature for 3-5min, adding 400 mu L of solution III, precipitating the protein at room temperature for 5min, then centrifuging at 13000rpm for 10min, carefully sucking the supernatant into a DNA-prep tube, centrifuging at 4000rpm for 1min, washing once with 500 mu L of buffer W1, washing twice with 500 mu L of buffer W2, centrifuging at 13000rpm for 1min, adding 40-50 mu L of TE to the bottom of the column, standing at room temperature for 1min, centrifuging at 13000rpm for 1min, and obtaining the centrifuged liquid as plasmid DNA solution.

8. Identification of recombinant plasmids

The obtained recombinant plasmid was sent to Shanghai Union Gene company for sequencing.

And II, obtaining a result.

1. And (5) converting the result.

The ligated target fragment was ligated to pET-28a (+) Fc plasmid, white colony mini-plasmid was picked and double digested, and the resulting DNA bands were all as expected, as shown in FIG. 1.

2. Sequencing results of recombinant plasmids

The obtained recombinant plasmid is sent to Shanghai combined gene company for sequencing, and the result shows that the sequence reading frame and the sequence direction are correct, which indicates that the target gene is correctly inserted into the expression plasmid.

Example 2

Expression and purification of recombinant protein in host bacteria

Firstly, a method is provided.

1. Transformation of

Coli competent BL21(DE3) was transformed with positive recombinant plasmid p by heat shock and cultured on LB resistant (containing 50. mu.g/ml ampicillin) agar plates at 37 ℃ for 12-16 h. The recombinant plasmid-transformed bacteria of the individual strains were picked up, inoculated into 10ml of LBG medium containing 50. mu.g/ml ampicillin, and shake-cultured at 37 ℃ to OD₆₀₀Reaching 0.4-0.6, adding IPTG until the final concentration is 0.3mmol/L, and continuously culturing at 200rpm for 3.5 h.

2. Inducible expression

Centrifuging the above induction product 1ml at 5000rpm to induce bacterial culture, discarding supernatant, adding 100 μ L PBS into the precipitate, mixing, adding 100 μ L2 xSDS loading buffer, boiling for 5min, centrifuging at 8000rpm at 4 deg.C for 5min, and collecting supernatant 20 μ L for SDS-PAGE electrophoresis.

3. Fusion protein expression analysis

After the induced bacteria were centrifuged at 6000rpm for 5min, please discard, and the cells were suspended in 1/10V PBS, sonicated at 300W for 5sec, 10sec, 50 times. After sonication, centrifugation was carried out at 12000rpm for 10min, and the supernatant and the precipitate were subjected to SDS-PAGE, respectively.

4. Washing and denaturation of inclusion body recombinant protein

(1) Washing: the target protein mainly existing in the form of inclusion body is resuspended by lysis buffer (50mmol/L Tris-HCl, pH8.0, L mmol/L EDTA, 100mmol/1L NaCl), and is primarily purified after washing by 2% Triton X/100 (polyethylene glycol octyl phenyl ether), 0.2% sodium deoxycholate, lmol/L urea.

(2) Denaturation: the initially purified inclusion bodies were denatured and solubilized with 8mmol/L urea (25mmol/L Tris-HCl, pH8.0, 20mmol/L DTT) for 6 h.

Purifying target protein by using SP-sepharose cation chromatographic column

(1) An SP-sepharose cation column (1ml) was attached to the FPLC and the column was washed with 10ml distilled water. The aim was to flush the column with 20% ethanol.

(2) The column was equilibrated with 5-10 column volumes of starting buffer solution (25mmol/L Tris-HCl, pH8.0, 0.2% dimercaptoethanol, 8mol/L urea). The flow rate was l ml/min.

(3) The sample is taken up by a syringe or a peristaltic pump at a flow rate of 1 ml/min. The passing peak was collected.

(4) Wash with 5-10 column volumes of starting buffer to 280nm absorbance to baseline level.

(5) Eluting with 10-25 column volumes of elution buffer (25mmol/L Tris-HCl, pH8.0, 8mol/L urea, 1mol/L NaCl) in linear gradient, and collecting each peak in tube.

(6) Elution buffer eluted for another 10 column volumes.

(7) The fractions of the target protein were determined by 12% SDS-PAGE electrophoresis.

(8) And (3) dialysis: fractions containing the protein of interest were dialyzed against the starting buffer for 48h, replacing the dialysate every 8 h.

SDS-PAGE detects the eluted sample, Coomassie brilliant blue stains, and a decolorant solution is used for taking pictures after decoloration.

6. Determination of the content of purified protein of interest

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

7. Renaturation of recombinant proteins

Renaturation by dilution under different oxidation conditions

(1) Redox agents:

adding DTT into the purified protein buffer solution to a final concentration of 10mmol/L, acting for 1h, and adding a reduction type: oxidized glutathione (2 mmol/L: 0.2mmol/L) was allowed to act on lh. According to the results of protein quantification, diluted to 0.5mg/ml with 4mol/L urea, and dialyzed and renatured with urea solution (containing reduced oxidized glutathione at the same concentration) at gradually decreasing concentration (4mol/L-2mol/L-0 mol/L).

(2) Air oxidation renaturation under alkaline conditions:

the purified P-5m-Fc fusion denatured protein was dialyzed into alkaline 8mol/L urea buffer (25mmol/L Tris-HCl, pH10.0), DTT was added to a final concentration of 10mmoI/L for L hours, the mixture was exposed to air and stirred overnight, and according to the protein quantification results, the mixture was diluted to 0.5mg/ml, and then dialyzed into urea buffer (25mmol/L Tris-HCl, pH7.2) with gradually decreasing concentration (4mol/L-2mol/L-0 mol/L).

And II, obtaining a result.

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

Recombinant plasmids with correct sequencing were transformed into E.coli BL21(DE3), single colonies were picked, activated and then shake-cultured at 37 ℃ until OD600 was about 0.6-0.8. Adding IPTG to 0.5mmol/L, inducing expression for 4 hr, collecting bacteria, ultrasonic crushing bacteria, and centrifuging to obtain supernatant and precipitate. The results of SDS-PAGE of the whole cells, the supernatant and the pellet are shown in FIG. 2, FIG. 3 and FIG. 4, indicating that the P-5m-Fc fusion protein is mainly expressed in the form of inclusion bodies and has a molecular weight of about 34 kD.

2. Washing and dissolving of recombinant protein inclusion body

The inclusion body mainly comprises recombinant protein and some thallus impurities, including RNA polymerase, bacterial membrane protein, rRNA and plasmid DNA.

The formation of inclusion bodies facilitates the isolation and purification of recombinant proteins and the stabilization of proteins, but since inclusion bodies are misfolded forms of proteins and often inactive, they must be denatured and solubilized and renatured under appropriate conditions to refold into active protein molecules with the correct conformation. Generally, the inclusion bodies are solubilized by washing with low concentrations of denaturants and detergents to remove contaminating proteins from the surface of the inclusion bodies. The washing conditions are best to remove as much of the contaminating protein as possible without dissolving the inclusion bodies. After groping of different washing conditions and concentrations, the series of washing conditions of 2% Triton X-100, 0.2% sodium deoxycholate and L mol/L urea were finally determined, and the inclusion bodies were primarily purified, as shown in FIG. 5.

3. SP-sepharose cation column chromatography purification

The denatured fusion protein was further purified by cation column chromatography, and at pH8.0, the fusion protein was bound to a cation resin and eluted with a buffer containing NaCI, as shown in FIG. 6.

Example 3

Activity assay of P-5m-Fc fusion proteins

Western blotting test

Protein bands (Fc and P-5m-Fc fusion protein) analyzed by SDS-PAGE were transferred to PVDF membrane, blocked with 1% BSA at 4 ℃ overnight, washed 3 times with 0.05% PBST for 10min each, and added with 2000^×Diluting the positive serum, allowing to act at 37 deg.C for 2h, washing with 0.05% PBST for 3 times, each for 10min, adding 25000^×The diluted rabbit anti-human IgG enzyme-labeled antibody is washed 3 times with 0.05% PBST for 10min each, and PIERCE luminescent substrate is added for 3min at room temperature, and the mixture is exposed in a dark room, developed and fixed, and the result is shown in FIG. 6.

The above results indicate that Fc was not bound to the P-5m polyclonal antibody, while P-5m-Fc was specifically bound to the P-5m polyclonal antibody.

Two, indirect ELISA assay

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

(1) Antigen preparation

The synthesis of the polypeptide prepared from the antigen adopts a 9-fluoromethoxycarbonyl (Fmoc) solid phase synthesis method to synthesize a polypeptide fragment, a high performance liquid phase method is used for purification, and a purified sample is analyzed by HPLC, so that the purity is more than 97%. The polypeptide is linked with carrier protein-hemocyanin.

(2) Animal immunization

250 mul of purified polypeptide antigen solution is mixed with Freund's complete adjuvant with the same volume, and the mixture is fully emulsified and then used for immunizing New Zealand white rabbits by a back multipoint injection method. Taking polypeptide antigen liquid after 2 weeks, adding equivalent volume Freund's incomplete adjuvant, performing immunization once every two weeks, performing immunization 5 times in the whole course, performing blood test on marginal veins 5-7 days after the last immunization, performing carotid bleeding after the serum reaches the ideal titer by an indirect ELISA method, and collecting rabbit serum.

(3) Immune rabbit serum collection and purification

Rabbit serum was collected by carotid artery bleeding.

Preliminary purification of rabbit serum by salting out:

A. 10mL of serum was mixed with physiological saline in equal volume, saturated ammonium sulfate was added dropwise with stirring to a final concentration of 50%, and the resulting mixture was left overnight at 4 ℃ to precipitate the protein sufficiently.

B. Centrifuging at 3000rpm/min for 20min, and discarding the supernatant. Dissolving the precipitate in 10mL of normal saline, adding saturated ammonium sulfate dropwise while stirring to make the final concentration 33%, and precipitating at 4 deg.C for more than 3 h.

C. Centrifuging at 3000rpm/min for 20min, and discarding the supernatant.

D. Dissolving the obtained precipitate to 5mL by PBS, putting into a dialysis bag, dialyzing for 1-2 d by PBS with the volume more than 20 times of the precipitate at 4 ℃, and changing PBS for a plurality of times during dialysis until the external liquid of the naphthalene reagent for detection is yellow.

E. The primarily purified rabbit serum is separated and purified by a C18 reverse phase chromatographic separation column and then is frozen and dried by suction. (affinity purified antibody IgG lyophilized powder quantitative, 1 mg/branch, -20 ℃ storage).

(4) Indirect ELISA method for determining serum titer

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

A. Coating: dissolving the synthesized polypeptide in 0.1mol/L carbonate buffer solution (pH 9.6) to prepare 100 mu g/ml antigen coating solution, coating an enzyme label plate by 100 mu L, simultaneously setting up a negative control (1: 200 diluted normal rabbit serum) and a blank control (no primary antibody), coating for 3h at 37 ℃ in 3 repeated wells with 100 mu L/well;

B. washing: washing with 250 μ L/hole washing solution for 5min for 3 times;

C. and (3) sealing: sealing with 200 mu L/hole sealing liquid at 37 ℃ for 2 h;

D. washing: washing with 250 μ L/hole washing solution for 5min for 3 times;

E. adding a primary antibody: adding 1: incubating rabbit immune serum diluted by 200 at the temperature of 37 ℃ for 1h, wherein the concentration of the rabbit immune serum is 100 mu L/hole;

F. washing: washing with 250 μ L/hole washing solution for 5min for 3 times;

G. adding a secondary antibody: adding 10000 goat anti-mouse IgG-HRP (1: 10000) per hole, and incubating at 37 ℃ for 1 h;

H. washing: washing with 250 μ L/hole washing solution for 5 times, each time for 5 min;

I. color development: adding OPD using solution with the concentration of 100 mu L/hole, and developing for 10-20 min in a dark place;

J. and (3) terminating the reaction: 2mol/L sulfuric acid, 50. mu.L/well.

The value A492 is measured by a microplate reader, and the positive is determined by (detected sample value-blank value)/(negative control value-blank value), namely P/N is more than or equal to 2.1.

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

The result shows that the P-5m peptide segment has good immunogenicity, the in vivo antibody titer begins to rise 2 weeks after the first immunization, namely 3 weeks, the rabbit antiserum and the polypeptide antigen have strong positive reaction after the 5 times of active immunization, namely 9 weeks, and the rabbit serum of a control group does not have immunoreaction with the polypeptide. The serum obtained after the rabbit is immunized is diluted in a multiple ratio, the OD value of a reaction hole is reduced along with the increase of the dilution of the serum, the positive is that P/N is not less than 2.1, the indirect ELISA measurement titer is more than 1: 160000, and the high-titer polyclonal antibody is obtained.

3. Detection of

The purified P-5m-Fc fusion proteins are coated with 10 μ g/mL ELISA plates at 4 ℃ overnight, then washed 3 times with 0.05% PBST for 3min each, blocked by adding 1% BSA at 37 ℃ for 2h, washed 3 times with 0.05% PBST for 3min each, and added with 50 XdilutedReacting the negative and positive serum at 37 deg.C for 1h, washing with 0.05% PBST for 3 times, each time for 3min, adding 10000^×Diluting rabbit anti-human IgG enzyme-labeled antibody, acting at 37 deg.C for 0.5h, washing with 0.05% PBST for 3 times, each time for 3min, adding substrate OPD, acting at 37 deg.C for 15min, 2mol/L H₂SO₄Upon termination, OD490 values were read using an ELx800 microplate reader.

The results indicate that high titers of P-5m polyclonal antibodies were obtained.

Functional study of anti-tumor metastasis of P-5m-Fc peptide body

1. Expression and purification of Fc control protein

In order to confirm that the peptide part plays a role and simultaneously expresses a control Fc protein, the constructed pET-28a-Fc fusion expression plasmid is transformed into BL21(DE3), IPTG induction expression (figures 2 and 3), thalli are collected, ultrasonic bacteria breaking is carried out, an inclusion body part is collected by centrifugation, washing and urea solution denaturation and dissolution are carried out as the same as the fusion protein, and purification is carried out.

2. Inhibition of HCCLM3 cell invasion matrix membrane by fusion protein

(1) Method of producing a composite material

A. Matrigel-free trans cell was coated with basement membrane: the Matrigel was diluted to 200. mu.g/ml on ice with serum-free RPMI 1640 medium, 100. mu.l of the mixed liquid was added to each upper chamber, and CO was added₂After the coagulation in the constant temperature incubator for 30min, the excess liquid was sucked off.

B. Hydrated basement membrane: adding 500 μ l serum-free RPMI 1640 culture medium into upper and lower chambers, respectively, adding CO₂Hydrating in constant temperature incubator for 30 min.

C. Preparing a cell suspension: the HCCLM3 cells in logarithmic growth phase were digested, the original culture medium was discarded, and the cells were diluted to 5X 10 with serum-free medium containing 10mg/ml BSA⁵cells/ml, and P-5M was added to final concentrations of 0. mu.M, 10. mu.M and 100. mu.M.

D. Inoculating cells: 200. mu.l of each cell suspension was inoculated into the upper chamber of a transwell, 500. mu.l of RPMI 1640 medium containing 10% FBS was added into the lower chamber, and CO was added₂Culturing for 24h in a constant temperature incubator.

E. Fixing: the upper chamber liquid was aspirated, the cells on the membrane and Matrigel gel were gently wiped off with a cotton swab, the cells under the membrane were fixed with ice-cold methanol for 10min, and air-dried.

F. Dyeing: staining with 0.1% crystal violet at room temperature for 15min, washing with sterile water for 3 times, and air drying.

G. The cells were photographed under an inverted microscope (400 ×), and the number of cells in 5 fields per well was counted to compare the difference in the invasiveness of the cells in each group. The experiment was repeated three times.

(2) Results

Through the transwell invasion experiment, the inhibition effect of the P-5m-Fc fusion protein on the migration of the hepatoma cells after 48 hours of action is examined.

As shown in FIGS. 7-8, the results of the invasion test of HCCLM3 cells showed that 10 μ M P-5m-Fc treated group had 31.6% fewer transmembrane cells than the control group, 100 μ M P-5m treated group had 43.4% fewer transmembrane cells than the control group, and the difference was statistically significant (P.ltoreq.0.05); the number of transmembrane cells after the treatment of 100 mu M P-5M-Fc and the P-5M polyclonal antibody is 11.9 percent less than that of a control group, and the difference has no statistical significance. The invasion experiment further defines the influence of P-5m on the invasion of tumor cells after the antibody is blocked, and the result shows that the antibody can block the inhibition capacity of P-5m-Fc on the invasion of tumor cells.

Moreover, as can be seen from the experimental results, the number of the transmembrane cells of the 10 mu M P-5m-Fc treatment group is 13.0 percent less than that of the transmembrane cells of the P-5m treatment group with the same dose, and the difference has statistical significance (P is less than or equal to 0.05); the number of transmembrane cells in the 100 mu M P-5m-Fc treatment group was 12.3% less than that in the P-5m treatment group at the same dose, and the difference was statistically significant (P.ltoreq.0.05).

The results show that the P-5m-Fc fusion protein after expression and purification has better activity for resisting tumor cell invasion, and the effect for resisting tumor cell invasion is better than that of the P-5m octapeptide.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A P-5m-Fc fusion protein, which is characterized in that the protein is formed by connecting a functional unit and an Fc part of human immunoglobulin IgG1, wherein the functional unit is formed by connecting an amino acid sequence shown in SEQ ID NO.1 and a linker, the linker is a polypeptide formed by six sequentially connected glycines, at least one functional unit is arranged in the P-5m-Fc fusion protein, and when a plurality of the functional units are arranged in the P-5m-Fc fusion protein, the functional units are sequentially connected and then connected with the Fc part of the immunoglobulin;

the P-5m-Fc fusion protein is prepared by the following method:

(1) constructing an expression gene, inserting the expression gene into a vector, and constructing a recombinant expression vector;

the sequence of the expression gene is as follows: is composed of a nucleotide sequence of a functional unit of a nucleotide sequence shown as SEQ ID NO.2 and an Fc part nucleotide sequence of coded immunoglobulin;

(3) transferring the positive expression vector into an expression cell, and performing induced expression on the expression gene sequence in the step (1);

(4) and (3) purification: collecting thalli after induction expression, suspending the thalli by using a phosphate buffer solution, carrying out ultrasonic crushing and centrifuging to obtain P-5m-Fc fusion protein expressed by inclusion bodies; resuspending the inclusion body by a lysis buffer solution, wherein the lysis buffer solution comprises 50mmol/L Tris-HCl with pH8.0, L mmol/L EDTA, 100mmol/1L NaCl, and then washing by 2% volume percentage of Triton X/100, 0.2% mass percentage of sodium deoxycholate and 1mol/L urea for preliminary purification;

the condition of the redox agent renaturation is as follows: adding DTT into the purified P-5m-Fc fusion protein buffer solution to a final concentration of 10mmol/L, then adding reduced glutathione and oxidized glutathione to concentrations of 2mmol/L and 0.2mmol/L respectively, diluting the P-5m-Fc fusion protein to 0.5mg/ml by using 4mol/L urea, dialyzing for renaturation by using urea buffer solutions with concentrations of 4mol/L, 2mol/L and 0mol/L, wherein the urea solution contains reduced glutathione and oxidized glutathione with the same concentration;

the air oxidation renaturation condition under the alkaline condition is as follows: dialyzing the purified P-5m-Fc fusion protein into 25mmol/L Tris-HCl buffer solution with 8mol/L urea content and pH value of 10.0, adding DTT to the final concentration of 10mmol I/L, exposing the solution to the air, stirring the solution overnight, diluting the P-5m-Fc fusion protein to 0.5mg/ml, and dialyzing the solution for renaturation by urea buffer solution with the concentration of 4mol/L, 2mol/L and 0 mol/L;

2. A method of preparing a P-5m-Fc fusion protein according to claim 1, comprising the steps of:

(1) constructing the expressed gene according to claim 1, wherein the sequence of the expressed gene is as follows: is composed of a nucleotide sequence of a functional unit of a nucleotide sequence shown as SEQ ID NO.2 and an Fc part nucleotide sequence of coded immunoglobulin; inserting the expression gene into a vector to construct a recombinant expression vector

3. The method of producing a P-5m-Fc fusion protein according to claim 2,

in the step (1), a recombinant expression vector is constructed by the following method: firstly, obtaining a gene fragment with a base sequence component of SEQ ID NO.2, carrying out double digestion on pET-28a (+) Fc plasmid by using BamH I and Nde I, recovering a digested vector fragment, and inserting the gene fragment of SEQ ID NO.2 into the vector through DNA ligase to obtain a recombinant plasmid, namely a recombinant expression vector;

transferring the ligation product into escherichia coli competence DH5 α by a heat shock method, coating the escherichia coli competence DH5 α on an LB agar culture medium containing X-gal, isopropyl- β -D-thiogalactoside and ampicillin, and screening to obtain a positive plasmid, namely the positive expression vector;

4. The method of producing a P-5m-Fc fusion protein according to claim 3,

in the step (2), the ligation product is transferred into escherichia coli competent DH5 α by a heat shock method, and is coated on an LB agar culture medium containing X-gal, isopropyl- β -D-thiogalactoside and ampicillin, after culture for 12-16h, a single colony is picked up and inoculated on an LB culture medium containing 50 mug/ml ampicillin, and positive plasmids are obtained by screening;

in the step (3), the positive plasmid obtained in the step (2) is transferred into escherichia coli E.coli BL21(DE3) competent cells through a heat shock method, and the competent cells are coated on an LB agar culture medium containing 50 mu g/ml ampicillin and cultured for 12-16 h; single colonies were picked and inoculated to 50. mu.g/ml ammoniaIn LBG medium with benzyl penicillin, shake culture to OD₆₀₀Adding isopropyl- β -D-thiogalactoside to a final concentration of 0.2-0.6mmol/L when the concentration reaches 0.4-0.6, performing induction culture, centrifuging the culture, taking the precipitate, adding phosphate buffer solution and loading buffer solution, boiling, and centrifuging again to obtain the P-5m-Fc fusion protein.

5. Use of the P-5m-Fc fusion protein of claim 1 in the preparation of a medicament for inhibiting metastasis of hepatoma cells.