CN113956347B - IL-8 mutants and uses thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5421—IL-8
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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Abstract
The invention belongs to the technical field of protein engineering, and particularly relates to an IL-8 mutant and application thereof, wherein the IL-8 mutant is an amino acid sequence shown in SEQ ID No:1, the base sequence is shown as SEQ ID No:2, and the mutation site is L25R. The amino acid sequence of the IL-8 mutant is shown as SEQ ID No:5, which are useful as antigens for the preparation of IL-8 antibodies and affinity chromatography columns. When the IL-8 mutant is adopted to prepare the affinity chromatographic column for purifying the specific IL-8 antibody, the specific IL-8 antibody is easier to elute and separate on the premise of small change of the binding rate of the specific IL-8 antibody and the affinity chromatographic column, the purity of the purified antibody is high, and the recovery rate is greatly improved.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to an IL-8 mutant and application thereof.
Background
Interleukin 8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCl 8) is a chemokine produced by cell types that are primarily macrophages, epithelial cells, airway smooth muscle cells, endothelial cells, and the like. Endothelial cells store IL-8 secretion in vesicles, i.e., weibel-Palade cells, with many receptors on the surface membrane that bind IL-8, the most studied of which are the G-protein coupled receptors CXCR1 and CXCR2.IL-8 has a different affinity between the two receptors, CXCR1 > CXCR2, and is secreted through a cascade of biochemical reactions, an important mediator in the response of the innate immune system. IL-8 has two main functions: firstly, it induces chemotaxis in target cells, mainly on neutrophils, but also on other granulocytes, causing them to migrate towards the site of infection, stimulating it to exert phagocytosis; secondly, IL-8 is a potent promoter of angiogenesis. At the position ofIL-8 induces a series of physiological responses required for migration and phagocytosis in target cells, e.g. intracellular Ca 2+ Increased, exocytosis (e.g., histamine release), etc. In general, macrophages first recognize foreign antigens in the immune response system of the body and are therefore the first cells to release IL-8 to recruit other cells. Both monomeric and homodimeric forms of IL-8 are reported to be potent inducers of the chemokine receptors CXCR1 and CXCR2.IL-8 is thought to play a role in the pathogenesis of bronchiolitis, a common respiratory disease caused by viral infection. The genes of IL-8, a member of the CXC chemokine family and the other ten members of the CXC chemokine family, form a cluster in the region of chromosome 4 q. The research finds that IL-8 is closely related to the occurrence and development of various diseases, especially tumors, and the specific blocking of IL-8 and the receptor CXCR1/2 thereof is expected to become a potential treatment strategy of various diseases. Therefore, the detection of the expression of IL-8 in the organism has more and more important significance in clinical treatment detection.
Detection of IL-8 in humans requires the preparation of monoclonal antibodies of high affinity and purity. At present, the purification of the IL-8 monoclonal antibody at home and abroad mainly comprises an affinity chromatography column, a Protein A column, a Protein G column and the like coupled with the IL-8 antigen. The purification yield and the relative purity of the Protein A column and the Protein G column are lower, and the monoclonal antibody purified by the affinity chromatography column coupled with the IL-8 antigen has good specificity and relatively higher purity, but the elution of the antibody is very difficult and the yield is lower because the binding force of the IL-8 antigen affinity column and the antibody is strong.
Disclosure of Invention
The invention aims to solve the problems in the prior art, provides an IL-8 mutant and application thereof, and provides an IL-8 mutant with a modified protein structure so as to reduce affinity with an IL-8 monoclonal antibody.
The technical scheme of the invention is as follows:
according to the existing IL-8 sequence information, analyzing the IL-8 amino acid sequence, screening out the amino acid sites which have obvious influence on the IL-8 conformation, and carrying out amino acid mutation on the corresponding sites through primer design. The conformation of IL-8 protein changes, thereby affecting the binding force of IL-8 to the antibody. Experiments prove that through mutation of IL-8 protein and a large number of screening, the L25R mutation site at the C end can cause the attenuation of the affinity of IL-8 antigen and antibody, thereby facilitating the invention.
In one aspect, the invention provides an IL-8 mutant with an amino acid sequence shown in SEQ ID No:1, the base sequence is shown as SEQ ID No:2, wherein the mutation site of the IL-8 mutant is L25R. According to the invention, through an IL-8 protein mutation experiment and a large number of screening, the L25R mutation site at the C end is determined to be capable of causing the attenuation of the affinity of IL-8 antigen and antibody.
Further, the amino acid sequence of the IL-8 mutant is shown as SEQ ID No:5, SAKELRCQCIKTYSKPFHPKFIKERRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.
Further, the nucleotide sequence of the IL-8 mutant is shown as SEQ ID No:6, AGTGCTAAAGAACTTAGATGTCAGTGCATAAAGACATACTCCAAACCTTTCCACCCCAAATTTATCAAAGAACGGAGAGTGATTGAGAGTGGACCACACTGCGCCAACACAGAAATTATTGTAAAGCTTTCTGATGGAAGAGAGCTCTGTCTGGACCCCAAGGAAAACTGGGTGCAGAGGGTTGTGGAGAAGTTTTTGAAGAGGGCTGAGAATTCATAA.
Further, constructing a primer pair of the IL-8 mutant, wherein the nucleotide sequence of an upstream primer is SEQ ID No:3, i.e. 5' -GGAATTCGGATCCAGTGCTAAAGAACTTAG-3'; the nucleotide sequence of the downstream primer is SEQ ID No:4, i.e. 5' -ACGCCTCGAGTTATGAATTCTCAGCCCTC-3’。
The invention also provides application of the IL-8 mutant in preparing an IL-8 antibody.
Further, the IL-8 mutant acts as an antigen.
An affinity chromatographic column is used for purifying IL-8 antibodies, takes agarose gel particles as a carrier and is coupled with the IL-8 mutant provided by the invention.
The invention has the beneficial effects that:
the invention realizes the change of the protein structure of the IL-8 mutant through the mutation of the IL-8 protein, reduces the affinity between the IL-8 mutant and the antibody, and is suitable for preparing the IL-8 antibody with high purity; when the IL-8 mutant is adopted to prepare the affinity chromatographic column for purifying the specific IL-8 antibody, the specific IL-8 antibody is easier to elute and separate on the premise of small change of the binding rate of the specific IL-8 antibody and the affinity chromatographic column, the purity of the purified antibody is high, and the recovery rate is greatly improved.
Drawings
FIG. 1 is a graph showing the results of analysis of the purity of IL-8 mutant proteins provided by the invention.
Detailed Description
The technical solutions in the preferred embodiments of the present invention will be clearly and completely described below with reference to examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, materials, and the like employed, unless otherwise specified, are commercially available.
EXAMPLE 1 construction of IL-8 Gene mutation library
The amino acid sequence of the IL-8 antigen (purchased from Shanghai friendly Biotechnology Co., ltd.) is shown in Table SEQ ID No:1, i.e. SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS; the base sequence is shown in SEQ ID No:2, namely: AGTGCTAAAGAACTTAGATGTCAGTGCATAAAGACATACTCCAAACCTTTCCACCCCAAATTTATCAAAGAACTGAGAGTGATTGAGAGTGGACCACACTGCGCCAACACAGAAATTATTGTAAAGCTTTCTGATGGAAGAGAGCTCTGTCTGGACCCCAAGGAAAACTGGGTGCAGAGGGTTGTGGAGAAGTTTTTGAAGAGGGCTGAGAATTCATAA.
In order to solve the problem that the IL-8 antigen (the amino acid sequence of which is shown as SEQ ID No. 1 and the nucleotide sequence of which is shown as SEQ ID No. 2) has strong binding force with an antibody, the protein is subjected to a large number of mutation screening by a directed evolution technology, and a pair of PCR primers are optimized and designed by self:
the upstream primer is shown in a sequence table SEQ ID No:3, namely: 5' -GGAATTCGGATCCAGTGCTAAAGAACTTAG-3'; the downstream primer is shown in a sequence table SEQ ID No:4, namely: 5' -ACGCCTCGAGTTATGAATTCTCAGCCCTC-3’。
EXAMPLE 2 construction and fermentation of IL-8 mutant expression Strain
(1) Using IL-8 gene as template, using the above primer, adding 2X TransStart FastPfu PCR SuperMix to amplify the whole plasmid gene; the PCR reaction conditions were: denaturation at 94℃for 5min; then denaturation at 94℃for 30s, renaturation at 60℃for 30s, extension at 72℃for 5min,30 cycles, sufficient extension at 72℃for 10min, and preservation at 4 ℃. And after the PCR is finished, adding DMT enzyme to digest in vitro degradation non-mutant plasmid template, and recovering the target gene.
(2) The glue recovery mutant gene and pET-28a vector are digested with BamHI and XhoI, the same terminal is exposed, T4 ligase is connected at room temperature, DMT competent cells are transformed, LB+Kan plates are coated, and the mixture is cultured at 37 ℃ overnight; the next day, recombinant strains were obtained by screening.
(3) The monoclonal colonies obtained by the screening are picked from the culture plate and inoculated into 10mL of LB+Kan culture medium, and after the culture at 37 ℃, plasmids are extracted and sent to a sequencing company for detection.
(4) Successful plasmid transformation was detected in BL21 (DE 3) competence, coated with LB+Kan plates and incubated overnight at 37 ℃; selecting monoclonal colony, inoculating to 5mL LB+Kan culture medium, culturing at 37deg.C, inoculating to 500mL LB+Kan culture medium, and culturing at 37deg.C to OD 600nm =0.6 to 0.8, cooled to 16 ℃, and induced overnight with 0.5mM IPTG.
The amino acid sequence of the IL-8 mutant protein is shown in a sequence table SEQ ID No:5, the mutation site of the IL-8 mutant protein is L25R, namely: SAKELRCQCIKTYSKPFHPKFIKERRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.
The coding gene sequence of the IL-8 mutant protein is shown as a sequence table SEQ ID No. 6, namely: AGTGCTAAAGAACTTAGATGTCAGTGCATAAAGACATACTCCAAACCTTTCCACCCCAAATTTATCAAAGAACGGAGAGTGATTGAGAGTGGACCACACTGCGCCAACACAGAAATTATTGTAAAGCTTTCTGATGGAAGAGAGCTCTGTCTGGACCCCAAGGAAAACTGGGTGCAGAGGGTTGTGGAGAAGTTTTTGAAGAGGGCTGAGAATTCATAA.
EXAMPLE 3IL-8 mutant purification
Buffer a:50mM PBS,500mM NaCl
Buffer B:50mM PBS,500mM NaCl,500mM imidazole
The purification steps are as follows:
centrifuging the bacteria liquid subjected to induced expression at 8000rpm for 5min, re-suspending the bacteria body by using the buffer solution A, performing ultrasonic crushing for 30min, centrifuging at 12000rpm for 30min, and collecting supernatant; adding the collected supernatant into Ni-NTA for binding for 1h, and eluting the target protein in steps by using buffer solution A and buffer solution B prepared by using buffer solutions with imidazole concentration of 10mM,20mM,50mM and 200 mM; starting a peristaltic pump, setting the flow speed to be 30rpm, simultaneously opening a protein detector, and flushing the protein detector to a baseline of the detector by using buffer solutions with gradients until the protein detector is stable; collecting various gradient eluted protein samples, and taking a small amount of samples to prepare samples required by SDS-PAGE; and collecting target protein, concentrating, and measuring the protein concentration. The results of the analysis of IL-8 antibody and purified and collected IL-8 mutant protein purity are shown in FIG. 1.
EXAMPLE 4IL-8 mutant affinity column coupling Process
The coupling process comprises the following steps:
1) Activated sepharose4b resin
Weighing 1.00g of sepharose4b resin, putting the sepharose4b resin into a beaker, adding 50mL of 1mM hydrochloric acid, swelling for 5min at room temperature, transferring into a suction filtration bottle, suction-filtering, directly adding 50mL of 1mM hydrochloric acid, immersing for 5min at room temperature, suction-filtering, and repeating for 5 times; (multiple times) 15-20 mL of 1mM hydrochloric acid was added and the resin was pipetted into a small triangular flask for ready coupling.
2) Mutant protein preparation
IL-8 mutant proteins were pipetting to 100mM NaHCO 3 (pH 8.3, containing 0.5M sodium chloride) at a concentration of 10mg/mL.
3) Coupling mutants
The resin in the small triangular flask of the above step 1) was naturally settled and the supernatant was aspirated (200. Mu.L gun was used to aspirate as much supernatant as possible after tilting the flask) before additionThe treated IL-8 mutant was supplemented with 1mLNaHCO 3 The buffer solution is oscillated on a 50rpm shaking table at 25 ℃ for 1-2 h.
4) Determination of protein concentration
Transferring the coupling body into a centrifuge tube, centrifuging at 8000rpm and 4 ℃ for 10min, taking supernatant, measuring volume, detecting protein content, and calculating the coupling rate.
5) Antigen coupling body cleaning treatment
Washing the coupling body with 50mL of 0.1M sodium bicarbonate (pH 8.3 and 0.5M sodium chloride), filtering, washing with 50mL of sodium bicarbonate, filtering, adding 100mL of 0.1M Tris-HCl buffer solution (pH 8.0), blocking the lower end of the filtering bottle with a preservative film, and soaking and sealing for 2h at room temperature;
after the sealing, the sealing solution is filtered out by pumping, and is alternately washed by 50mL of 0.1M sodium acetate-acetic acid buffer solution with the pH of 4.0 and 0.1M, pH 8.0.0 Tris-HCl buffer solution (containing 0.5M sodium chloride) respectively, and is filtered by pumping again for four times; finally, 50mL of PBS with the concentration of 0.02M and the pH of 8.0 is added for washing and suction filtration, and the steps are repeated for 3 times.
6) Column
Mixing with 10mL of 0.02M PBS, taking out, loading into column, naturally precipitating, tightening the column, washing with 0.02M PBS with pH of 7.4, and checking whether omission occurs;
before loading the column, 0.1mL of coupling heavy suspension can be taken for BCA measurement, and deeper blue color is displayed in the sediment, which indicates that the antigen is normally coupled during coupling and sealing and flushing.
Test example 1 comparison of IL-8 antibody purification methods
The method comprises the steps of utilizing human IL-8 protein to stimulate mice, hybridoma cells, collecting ascites, purifying antibodies and the like, preparing the IL-8 monoclonal antibody, designing and verifying three experimental methods according to the characteristics of the IL-8 antibody and starting from the purity, recovery rate, stability and the like of the antibody, and comparing the purification schemes.
Method (1): IL 8 antibody purification by protein G affinity chromatography column
Method (2): IL-8 antigen affinity chromatography column for purifying IL-8 antibody
Method (3): IL-8 antigen mutant affinity chromatographic column for purifying IL-8 antibody
Balance: washing protein G chromatographic column, IL-8 antigen affinity column and IL-8 antigen mutant affinity column with 20mL pure water, and washing three chromatographic columns with 20mL binding buffer (0.02M PBS, pH 8.0) at a rate of 1 mL/min;
loading: diluting the sample by 5 times by using a binding buffer solution, and sterilizing by using a 0.22um sterilizer; loading by using a syringe, and collecting penetrating fluid;
eluting: flushing the column with binding buffer until the protein detector baseline is unchanged, and discarding the effluent; washing the column with elution buffer (0.1 Mglycine-HCl, pH 2.8) until the protein detector baseline is unchanged, and collecting the effluent (i.e., the eluate); the collected target protein eluent is subjected to SDS-PAGE and Western-Blot, and antibody purity is analyzed by Bandscan scanning. As shown in Table 1, IL-8 antibody purity was greater than 95% by IL-8 antigen mutant affinity chromatography column using Bandscan scanning analysis software.
Monoclonal antibody affinity constant assay
Affinity constants represent how tightly an antibody binds to an antigen, and are one of the important parameters for determining the properties of an antibody, as well as an important indicator of the stability of a monoclonal antibody. The target proteins eluted by the three methods were subjected to SDS-PAGE and Western-Blot verification and then subjected to concentration measurement, and the measurement results are shown in Table 1 below.
(two) non-competitive ELISA determination of relative affinity constant of mAb
The experimental procedure was as follows:
(1) the ELISA plate is coated with three concentrations of human IL-8 protein of 20 mug/mL, 10 mug/mL and 5 mug/mL respectively, 100 mug/hole is carried out at 4 ℃ overnight;
(2) washing the plate, PBST 200 mu L/hole, washing 3 times, 5 min/time;
(3) closing: incubation with 3% BSA as blocking solution, 110. Mu.L/well, at 37℃for 2h;
(4) washing the plate: and (2);
(5) an antibody: adding a gradient diluted monoclonal antibody, 100 mu L/hole, and incubating for 2h at 37 ℃;
(6) washing the plate: and (2);
(7) and (2) secondary antibody: 1: diluting the enzyme-labeled secondary antibody by 3000, 100 mu L/hole, and incubating for 45min at 37 ℃;
(8) washing the plate: and (2);
(9) color development: adding substrate use solution, 100 mu L/hole, and keeping the temperature at 37 ℃ and developing for 10min in dark;
terminating: 50. Mu.L of stop solution was added to each well, and Abs values were measured at 450nm, and the measurement results are shown in Table 1 below.
(III) monoclonal antibody stability test
Also, binding force between antibody and antigen was measured before and after 3 months of cryopreservation by indirect ELISA, and OD was measured 450nm And comparing the differences between the light absorption values.
Table 1 results of comparative experiments of three antibody purification methods
| Method | Purity of | Recovery rate | Affinity constant of monoclonal antibody | Monoclonal antibody stability (Abs) |
| protein G affinity chromatography purification | 42.1％ | 73％ | 1.35×10 8 L/mol | 0.307 |
| IL-8 antigen affinity chromatography purification | 96％ | 38％ | 8.21×10 8 L/mol | 0.543 |
| IL-8 antigen mutant affinity chromatography purification | 95％ | 74％ | 7.43×10 8 L/mol | 0.521 |
As can be seen from the above table, three purification methods can obtain active IL-8 antibodies, wherein the purity and activity of the antibodies purified by protein G affinity chromatography are low; the recovery rate of the antibody purified by IL-8 antigen affinity chromatography is low; the IL-8 antigen mutant affinity chromatography purified antibody has the same activity and stability as the IL-8 antigen affinity chromatography purified antibody, but the recovery rate is greatly improved.
The foregoing description is only a preferred embodiment of the present invention and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, variation, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Qingdao Resmalls biotechnology Co.Ltd
<120> IL-8 mutant and use thereof
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Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro
1 5 10 15
Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro
20 25 30
His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu
35 40 45
Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys
50 55 60
Phe Leu Lys Arg Ala Glu Asn Ser
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<213> Artificial sequence (Artificial Sequence)
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agtgctaaag aacttagatg tcagtgcata aagacatact ccaaaccttt ccaccccaaa 60
tttatcaaag aactgagagt gattgagagt ggaccacact gcgccaacac agaaattatt 120
gtaaagcttt ctgatggaag agagctctgt ctggacccca aggaaaactg ggtgcagagg 180
gttgtggaga agtttttgaa gagggctgag aattcataa 219
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ggaattcgga tccagtgcta aagaacttag 30
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His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu
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gtaaagcttt ctgatggaag agagctctgt ctggacccca aggaaaactg ggtgcagagg 180
gttgtggaga agtttttgaa gagggctgag aattcataa 219
Claims (7)
1. An IL-8 mutant characterized by having an amino acid sequence as set forth in SEQ ID No:1, the base sequence is shown as SEQ ID No:2, and the mutation site is L25R.
2. The IL-8 mutant of claim 1, wherein the amino acid sequence of the IL-8 mutant is as set forth in SEQ ID No: shown at 5.
3. The IL-8 mutant of claim 1, wherein the IL-8 mutant has a nucleotide sequence as set forth in SEQ ID No: shown at 6.
4. The IL-8 mutant of claim 1, wherein the primer pair of the IL-8 mutant is constructed, and the nucleotide sequence of the upstream primer is SEQ ID No:3, the nucleotide sequence of the downstream primer is SEQ ID No:4.
5. use of an IL-8 mutant according to any one of claims 1 to 4 for the preparation of an IL-8 antibody.
6. The use according to claim 5, wherein the IL-8 mutant is used as an antigen.
7. An affinity chromatography column for purifying an IL-8 antibody, wherein the affinity chromatography column uses agarose gel particles as a carrier, and is coupled with the IL-8 mutant according to any one of claims 1 to 4.
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| AT504685A1 (en) * | 2006-12-20 | 2008-07-15 | Protaffin Biotechnologie Ag | FUSION PROTEIN |
| CN108179149A (en) * | 2018-01-03 | 2018-06-19 | 青岛瑞斯凯尔生物科技有限公司 | S100B mutant and its application |
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