CN116987700A - Human interleukin-5 recombinant protein and preparation method thereof - Google Patents
Human interleukin-5 recombinant protein and preparation method thereof Download PDFInfo
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- CN116987700A CN116987700A CN202310857994.8A CN202310857994A CN116987700A CN 116987700 A CN116987700 A CN 116987700A CN 202310857994 A CN202310857994 A CN 202310857994A CN 116987700 A CN116987700 A CN 116987700A
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- human interleukin
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Classifications
-
- 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]
- C07K14/5409—IL-5
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/98—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving alcohol, e.g. ethanol in breath
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/5409—IL-5
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a human interleukin-5 recombinant protein and a preparation method thereof, wherein 4 segments of sequences capable of forming antigen epitopes are selected for continuous repeated expression twice to obtain nucleic acid molecules for encoding the antigen epitopes of the human interleukin-5 recombinant protein, recombinant vectors are constructed and efficiently expressed in an escherichia coli expression system, and the prepared target protein has higher activity; and the renaturation and purification process is screened and optimized, the protein yield is obviously improved, the protein biological activity is kept, the target protein with higher activity than the human interleukin-5 protein standard can be obtained through one-step column purification, and the method has important scientific significance and clinical application value.
Description
Technical Field
The invention relates to the technical field of recombinant protein expression and purification, in particular to a human interleukin-5 recombinant protein and a preparation method thereof.
Background
Human Interleukin-5 (Interleukin-5, IL-5) is a secreted cytokine first discovered by Takasu et al in 1980 and is produced primarily by activated Th2 cells (helper T cells 2), eosinophils and mast cells. Human interleukin-5 plays the most important role in eosinophil growth, maturation, differentiation, activation, and migration, and can prolong eosinophil survival by inhibiting apoptosis; human interleukin-5, alone or in combination with transforming growth factor B, may promote IgA production by activated B cells via a humoral immune response.
Epidemiological studies have shown that Th 2-type cytokine-related allergic diseases (such as asthma, atopic dermatitis, allergic diarrhea and other atopic diseases) have seen a general trend in recent years, characterized by the appearance of inflammation, i.e., significant infiltration of T cells and granulocytes (including mast cells, eosinophils and neutrophils). Human interleukin-5 produced by Th2 cells, as a key cytokine in the development of allergic inflammation, causes the accumulation of eosinophil excessive proliferation by binding to the heterodimeric human interleukin-5 receptor (IL-5R). Eosinophils play an important role in the protective immune response by producing major basic proteins, eosinophil Cationic Proteins (ECP) and eosinophil-derived neurotoxins (EDN), which however have the ability to lyse cells, causing tissue damage in the body. It has also been found in previous studies that eosinophils may be involved in tumor monitoring by flowing into tumor growth sites, such as during the development of lung cancer, melanoma, fibrosarcoma, and the like, and that eosinophil infiltration may inhibit tumor formation and aid in the clearance of metastatic lesions. Therefore, the method for detecting the human interleukin-5 in the blood of the patient has wide application prospect in diagnosis of inflammation or allergic diseases and prognosis of acute rejection or treatment of tumors.
The existing human interleukin-5 recombinant protein is mainly produced and obtained through an insect baculovirus system and a CHO system, the expression period of the insect baculovirus system and the CHO system is long, the time from obtaining an expression plasmid to finishing protein expression is 1 month or longer, and the human interleukin-5 recombinant protein obtained in the eukaryotic system is an active protein which is completely glycosylated. However, it has been shown that the glycosylation process of eukaryotic systems is not necessary for human interleukin-5 to possess biological activity, and that human interleukin-5 biological activity is mainly related to its C-terminal residue and dimer structure, so that human interleukin-5 recombinant proteins with complete activity can also be obtained using prokaryotic system expression.
Compared with eukaryotic system expression, prokaryotic system expression has the advantages of simple and quick operation flow, high expression yield, low cost, suitability for industrialization and the like. The human interleukin-5 recombinant protein can obtain complete activity without further processing modification of a eukaryotic system, so that prokaryotic expression is a mode which is more suitable for the rapid production of the human interleukin-5 recombinant protein. However, the existing prokaryotic expression human interleukin-5 recombinant Protein (such as FEBS Lett (1993) 27;331 (1-2): 49-52.doi:10.1016/0014-5793 (93) 80295-6./Protein Expr purification (1997) 11 (1): 86-94.doi: 10.1006/prep.1997.0785.) generally adopts the mode of expressing complete sequences and purifying by multiple steps, and compared with the natural Protein, the prepared human interleukin-5 recombinant Protein has no advantage in reactivity, complicated purification process, difficult purification, long period and high cost, and can obtain the target Protein with higher purity by generally needing 3-4 gel chromatographic columns for purification.
Therefore, it is highly desirable to find a method for preparing a recombinant protein of human interleukin-5 with higher activity, higher reactivity and easier purification, which is of great importance for quantitative detection of human interleukin-5 in disease diagnosis and treatment prognosis stages.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a human interleukin-5 recombinant protein and a preparation method thereof, and the nucleic acid molecule for encoding the epitope of the human interleukin-5 recombinant protein is obtained by selecting a proper sequence capable of forming the epitope for continuous expression, a recombinant vector is constructed and is efficiently expressed in an escherichia coli expression system, and the activity of the prepared target protein is higher; and the denatured cleaning solution and renaturation process are screened and optimized, the protein yield is obviously improved, the protein biological activity is kept, the target protein with higher activity than the human interleukin-5 protein standard can be obtained through one-step column purification, and the method has important scientific significance and clinical application value.
In one aspect, the present invention provides a nucleic acid molecule for encoding a human interleukin-5 recombinant protein, said nucleic acid molecule comprising Ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 sequences repeated twice, said Ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 having the nucleotide sequences as shown in SEQ ID NO 1-4, respectively.
According to NCBI functional network inquiry, the invention obtains mRNA sequence (SEQ ID NO: 7) of human interleukin-5, selects Ile20-Ser134 (SEQ ID NO: 8) sequence except signal peptide from the mRNA sequence of human interleukin-5, predicts epitope of human interleukin-5 based on parameters such as hydrophobicity, protein secondary structure, epitope determinant and the like, screens 4 segments (Ala 45-Cys63 (SEQ ID NO: 1), ser75-Thr82 (SEQ ID NO: 2), lys103-Arg110 (SEQ ID NO: 3), val124-Ser134 (SEQ ID NO: 4)) and can form epitope sequence for continuous expression. It was found that nucleic acid molecules constructed using these 4 sequences are capable of expressing proteins of interest with higher biological activity.
Because human interleukin-5 has a dimeric form, the invention also attempts to repeatedly express the 4-segment sequence twice by using the flexible connecting peptide, and researches prove that the biological activity of the target protein can be obviously improved by repeatedly expressing the sequence twice.
Further, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO. 5.
In another aspect, the invention provides a recombinant vector comprising a nucleic acid molecule as described above.
In the invention, the recombinant vector has multiple cloning sites, and is a molecular biological vector capable of regulating and controlling the expression of exogenous genes.
In some embodiments, the empty vector of the recombinant vector comprises pET21a (+).
In yet another aspect, the invention provides an E.coli expression system for transforming a recombinant vector as described above.
In some embodiments, the E.coli expression system comprises E.coli host bacteria that have been DE3 solubilized.
In some embodiments, the E.coli host strain is BL21 (DE 3).
In a further aspect, the present invention provides a human interleukin-5 recombinant protein encoded by a nucleic acid molecule as described above.
The human interleukin-5 recombinant protein is obtained by adopting the expression system of the escherichia coli.
Further, the recombinant protein has an amino acid sequence shown as SEQ ID NO. 6.
In still another aspect, the present invention provides a method for preparing a recombinant human interleukin-5 protein, which is obtained by constructing a recombinant vector comprising the nucleic acid molecule as described above, and expressing the recombinant vector in a prokaryotic system.
Further, the preparation method comprises the following steps:
(1) Inserting the nucleic acid molecule into an escherichia coli expression vector to obtain a recombinant vector;
(2) The recombinant vector is transformed into an escherichia coli expression system to obtain a bacterial solution;
(3) Adding denatured lysate into the thallus solution for cracking and centrifuging to obtain protein solution;
(4) Purifying the protein solution by a column, and eluting to obtain protein eluent;
(5) Adding renaturation solution into the protein eluent, ultrafiltering, concentrating and dialyzing to obtain the human interleukin-5 recombinant protein.
Further, the method comprises:
(1) Inserting nucleic acid molecules encoding human interleukin-5 recombinant proteins into an escherichia coli expression vector to obtain a recombinant vector;
(2) The recombinant vector is transformed into an escherichia coli expression system for induction culture;
(3) Collecting thalli of the escherichia coli expression system, re-suspending the thalli containing the denatured lysate, and obtaining protein dissolution liquid (inclusion body dissolution liquid) through crushing and centrifugation;
(4) Purifying inclusion body dissolution liquid by column to obtain protein eluent;
(5) Slowly adding the protein eluent into renaturation solution to fully renaturate the human interleukin-5 recombinant protein under the low temperature condition, and then concentrating and dialyzing to obtain the renaturated human interleukin-5 recombinant protein.
In some embodiments, the E.coli expression vector of step (1) comprises pET21a (+).
In some embodiments, the nucleic acid molecule of step (1) is inserted into the BamHI and XhoI restriction sites of an E.coli expression vector.
In some embodiments, the E.coli expression system of step (2) comprises a DE 3-solubilized E.coli host strain.
In some embodiments, the E.coli host strain is BL21 (DE 3).
In some embodiments, the method of converting of step (2) is chemical conversion.
Further, the denatured lysate of step (3) includes PBS (phosphate buffer), urea, imidazole, triton X-100 (polyethylene glycol octyl phenyl ether) and PMSF (phenylmethylsulfonyl fluoride).
Further, the denatured lysate of step (3) comprises PBS 0.005-0.015M, urea 6-10M, imidazole 4-6 mM, triton X-100 0.5-1.5% and PMSF 0.5-1.5 mM, pH 7.5-8.5.
In some embodiments, the denaturing lysate of step (3) is 0.01M PBS,8M urea, 5mM imidazole, 1% Triton X-100, 1mM PMSF (phenylmethylsulfonyl fluoride), pH8.0. The denatured lysate can dissolve human interleukin-5 recombinant protein in solution as much as possible and prevent the target protein from degrading.
In some aspects, the method of crushing of step (3) is ultrasonication.
Further, step (4) the column purification comprises denaturation washing and denaturation elution; the denatured cleaning solution can clean other proteins as much as possible, and only the target proteins are reserved on the column.
In some embodiments, the denaturing wash of step (4) comprises 0.005 to 0.015M PBS,6 to 10M urea, and 30 to 100mM imidazole, pH7.5 to 8.5.
In some embodiments, the denatured-cleaning solution of step (5) is: 0.01M PBS,8M urea and 80mM imidazole, pH8.0. Research proves that aiming at the human interleukin-5 recombinant protein, the denatured cleaning solution is more beneficial to reducing the content of the hybrid protein as much as possible under the condition of ensuring that the target protein is not lost compared with denatured cleaning solutions under other imidazole concentration conditions.
In some embodiments, the denaturing eluent from the denaturing elution of step (4) comprises 0.005 to 0.015M PBS,6 to 10M urea, and 400 to 600mM imidazole, pH7.5 to 8.5. The denaturing eluent can completely elute the human interleukin-5 recombinant protein from the affinity column.
In some embodiments, the method of column purification of step (4) comprises affinity chromatography.
In some embodiments, the affinity chromatography is performed using a nickel column.
Further, the renaturation solution in the step (5) contains Tris (Tris (hydroxymethyl aminomethane) with pH of 8.0.
In some embodiments, the renaturation solution of step (5) comprises 40-60 mM Tris, 0.4-0.6M NaCl, 0.2-0.4M Arg (L-arginine), 0.5-2.5 mM GSH/GSSG (reduced glutathione/oxidized glutathione), and 0.5-1.5 mM DTT (dithiothreitol).
In some embodiments, the renaturation solution of step (5) is: 50mM Tris, 0.5M NaCl, 0.3M Arg, 2mM/1mM GSH/GSSG and 1mM DTT, pH8.0. Research proves that the renaturation liquid is better than renaturation liquid under other pH conditions aiming at human interleukin-5 recombinant protein from the two aspects of protein yield and protein reactivity.
According to the invention, research proves that the preparation of renaturation solution by selecting a Tris buffer system with pH of 8.0 and adding NaCl and Arg into the Tris buffer system are higher in protein reactivity and higher in protein yield of the prepared human interleukin-5 recombinant protein.
Further, the dialysate of the dialysis of step (5) comprises Tris, naCl and arginine.
In some embodiments, the low temperature conditions described in step (5) are 0-4 ℃.
In some embodiments, the concentration conditions described in step (5) are 3kDa ultrafiltration tube at 4℃overnight at 5000g or PEG6000 embedded at 4 ℃.
In some embodiments, the dialysate of step (5) comprises 40-60 mM Tris, 0.4-0.6M NaCl, 0-0.2 Arg, pH 7.5-8.5.
In some embodiments, the dialysate of step (5) is pH8.0, 50mM Tris, 0.5M NaCl, 0.2M/0.1M/0MArg. The dialysate can ensure the stability of the recombinant human interleukin-5 protein in the dialysis process, reduce precipitation and improve the yield of the recombinant human interleukin-5 protein.
In yet another aspect, the present invention provides the use of a nucleic acid molecule comprising two repeated Ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 sequences, said Ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 having the nucleotide sequences as shown in SEQ ID NO. 1-4, respectively, for the preparation of a highly active human interleukin-5 recombinant protein.
In yet another aspect, the invention provides a use of a recombinant human interleukin-5 protein for quantitative detection of human interleukin-5.
Furthermore, the human interleukin-5 is quantitatively detected and can be used for diagnosis of inflammation or allergic diseases and prognosis of acute rejection or treatment of tumors.
The invention has the beneficial effects that:
1. provides a method for preparing highly reactive human interleukin-5 recombinant protein rapidly, economically and effectively by using a prokaryotic expression system;
2. according to mRNA sequence of human interleukin-5, selecting Ile20-Ser134 sequence except signal peptide, predicting antigen epitope of human interleukin-5 based on hydrophobicity, protein secondary structure, antigen determinant and other parameters, selecting 4 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) of sequence which possibly forms antigen epitope for continuous expression and repeatedly expressing twice by using flexible connecting peptide to obtain nucleic acid molecule for encoding antigen epitope of human interleukin-5 recombinant protein;
2. cloning the nucleic acid molecules into an escherichia coli expression vector to obtain a recombinant vector after artificial synthesis, and transfecting the recombinant vector into an escherichia coli expression system, so that the high-efficiency expression of the human interleukin-5 recombinant protein is realized, the target protein accounts for 40% -50% of the total protein, the conditions of denaturation, cleaning, renaturation and the like are optimized and regulated, 1.3mg of human interleukin-5 recombinant protein with high activity can be obtained by 100mL of bacterial liquid, and the antigen reactivity of the recombinant protein is superior to that of the human interleukin-5 recombinant protein expressing the complete sequence under the same protein concentration condition;
3. the high-purity human interleukin-5 recombinant protein with the purity of more than 95% can be obtained by only one-step purification;
4. the preparation method has the characteristics of simple steps, low cost, higher recovery rate and strong antigen reactivity, has important significance and wide prospect in diagnosing inflammatory diseases or allergic diseases and acute rejection or treating prognosis of tumors and quantitatively detecting human interleukin-5, and can be used for preparing standard substances and antigens for preparing antibodies of immune animals.
Drawings
FIG. 1 is a diagram showing SDS-PAGE results of a human interleukin-5 recombinant protein prepared from an original sequence, wherein the left diagram shows a diagram showing SDS-PAGE results of a human interleukin-5 recombinant protein prepared from an optimized sequence, the right diagram shows a diagram showing SDS-PAGE results of a human interleukin-5 recombinant protein prepared from an optimized sequence, lane 1 shows a sample before purification, lane 2 shows a post-column collection solution, lane 3 shows a washing collection solution, lane 4 shows an elution collection solution, and lane 5 shows a protein Marker;
FIG. 2 is a graph showing the predicted protein hydrophobicity of the amino acid sequence of human interleukin-5 according to example 3;
FIG. 3 is a graph showing the results of predicting the secondary structure of human interleukin-5 amino acid sequence in example 3;
FIG. 4 is a graph showing the results of epitope prediction performed on the amino acid sequence of human interleukin-5 in example 3;
FIG. 5 is a diagram of the construction of plasmid pET21a (+) of an E.coli expression vector.
Detailed Description
The invention will be described in further detail below with reference to the drawings and examples, it being noted that the examples described below are intended to facilitate an understanding of the invention and are not intended to limit the invention in any way. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Materials and reagents:
competent cells: coli BL21 (DE 3) competent cells were purchased from Tiangen Biochemical technology (Beijing) Inc.
The culture medium comprises the following components: yeast Extract Powder Tryptone is available from OXOID, naCl from national pharmaceutical Chemie Co., ltd, and ampicillin from Tiangen Biochemical technology (Beijing) Co.
Lysate, equilibrium solution, cleaning solution, eluent, renaturation solution components: urea, na2 hpo4.12h O, KH2PO4, naCl, KCl were purchased from national pharmaceutical chemicals company, inc., imidazole was purchased from Sigma, GSH, GSSG, arg, DTT was purchased from the division of bioengineering (Shanghai).
Biochemical reagent: pierce TM BCA Protein Assay Kit FastDiget BamHI, fastDiget XhoI from Thermo Fisher; high Affinity Ni-NTA Resin is available from Kirschner Biofuraw, inc TM Quick protein dye liquor is purchased from Shanghai Tencenters Co., ltd; 2×Loading buffer, 12% SDS-PAGE prefabricated protein gel, MOPS running buffer from Nanjing Ai Saiyi biological departmentTechnology Co., ltd.
The human interleukin-5 standard is purchased from a racing organism and is a self-contained standard in an immunofluorescence cytokine combined detection kit (registration certificate number: ganchi mechanical standard 20192400359).
Example 1: the invention provides the preparation of human interleukin-5 recombinant protein
The preparation process of the human interleukin-5 recombinant protein provided in the embodiment comprises the following steps:
1. design and synthesis of human interleukin-5 recombinant protein coding gene
The mRNA sequence of human interleukin-5 (NCBINCBI Reference Sequence accession number: NM_ 000879.3) disclosed by NCBI is taken as an optimization object, and the Ile20-Ser134 sequence is selected as a control sequence, wherein the sequence is shown as SEQ ID NO. 8; based on parameters such as hydrophobicity, protein secondary structure, antigenic determinant and the like, predicting the epitope of the human interleukin-5, selecting 4 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) of sequences which are likely to form the epitope, continuously expressing the epitope, repeatedly expressing the epitope twice by using flexible connecting peptide to obtain a nucleic acid molecule for encoding the epitope of the human interleukin-5 recombinant protein, wherein the sequence is shown as SEQ ID NO. 5, and performing manual synthesis.
The prepared nucleic acid molecule (SEQ ID NO: 5) encoding the human interleukin-5 recombinant protein epitope has the following sequence:
ATG-GCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCGGTGGTGGT GGTAGCGGTGGTGGTGGTAGCGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCTGA
including the 4-segment (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) sequences, expressed in duplicate, as follows:
2. acquisition of human interleukin-5 recombinant engineering bacteria
(1) Chemically synthesizing a gene encoding the human interleukin-5 recombinant protein (SEQ ID NO: 5) and adding BamHI and XhoI cleavage sites at both ends (chemical synthesis is performed at Kirschner Biotechnology Co., ltd.), and after the gene encoding the human interleukin-5 recombinant protein (SEQ ID NO: 5) is cleaved by restriction enzymes BamHI and XhoI, purifying and recovering by TaKaRa Fragment Purification Kit; inserting the recovered coding gene into pET21a (+) which is also digested by restriction enzymes BamHI and XhoI to construct a recombinant vector; the plasmid structure of the E.coli expression vector used is shown in FIG. 5.
(2) Slowly thawing competent cells of Escherichia coli BL21 (DE 3) on ice, adding about 40ng of recombinant vector, gently mixing, and continuing ice bath for 20min;
(3) The competent cells added with the recombinant vector are heated in a water bath kettle at 42 ℃ for 90s and rapidly transferred to ice for 2-5 min;
(4) Adding 500uL LB culture solution (without resistance) into competence containing recombinant vector in a sterile workbench, and culturing at low speed for 0.5h at 37 ℃;
(5) And (3) uniformly coating the culture solution on 200 mu L of LB solid medium with the resistance to the culture solution liquid ammonia in a sterile workbench by using a disposable sterile coating rod, and culturing the culture solution in a constant temperature incubator at 37 ℃ for 12-16 hours after inversion, wherein the bacterial colony growing in the next day is the human interleukin-5 recombinant engineering bacterium.
3. Expression of human interleukin-5 recombinant protein
(1) Monoclonal inoculating the grown human interleukin-5 recombinant engineering bacteria into an ampicillin-resistant LB culture medium, and culturing overnight at 37 ℃ and 220rpm to obtain seed solution;
(2) Adding the seed solution into an ampicillin-resistant 2YT liquid culture medium at a ratio of 1:100 for expansion culture, culturing at 37 ℃ and 220rpm until OD600 is approximately equal to 1, adding IPTG with a final concentration of 0.5mM for induction, and continuously culturing at 37 ℃ and 220rpm for 4 hours;
(3) After the completion of the culture, the cells were centrifuged at 12000rpm for 5 minutes, and the supernatant was discarded to obtain cells expressing human interleukin-5.
4. Purification and renaturation of human interleukin-5 recombinant protein
(1) Mixing wet thalli obtained by centrifuging 100mL of bacterial liquid with 10mL of denatured lysate (0.01M PBS,8M urea, 5mM imidazole, 1% Triton X-100, 1mM PMSF (phenylmethylsulfonyl fluoride), pH 8.0), adding PMSF with a final concentration of 1mM, mixing thoroughly, and placing in an ice-water mixture;
(2) Performing ultrasonic crushing by using an ultrasonic crusher, wherein the power is 300W,5s and 5s, and the total working time is 5min;
(3) Centrifuging the ultrasonic bacterial liquid at 12000rpm for 5min, reserving supernatant, and filtering by using a PVDF filter membrane with the concentration of 0.45 mu M;
(4) Filling 2mL of nickel column filler into a column, balancing the nickel column by using a denatured binding solution with the volume of 10 times of the column volume, slowly loading the filtered ultrasonic supernatant into the column, and collecting post-column liquid; in the embodiment, only one-step purification and column passing operation are needed;
(5) Eluting with 5 times of column volume of denatured washing liquid (0.01M PBS,8M urea and 80mM imidazole, pH 8.0) and 2 times of column volume of denatured eluent (0.01M PBS,8M urea and 500mM imidazole, pH 8.0), and collecting washing liquid and eluent to obtain purified human interleukin-5 protein;
(6) Slowly dripping the nickel column eluent into protein renaturation solution (50 mM Tris, 0.5M NaCl, 0.3M Arg, 2mM/1mM GSH/GSSG and 1mM DTT, pH8.0) containing 0.3MArg dropwise according to a dilution ratio of 1:50, and continuously stirring at low temperature for 36h;
(7) Concentrating the fully renatured protein liquid by using a 3KD ultrafiltration tube, wherein the centrifugation condition is 4 ℃ and 5000g;
(8) The protein renaturation concentrated solution is subjected to gradient substitution in a dialysis solution (pH 8.0, 50mM Tris and 0.5M NaCl) containing 0.2M, 0.1M and 0M Arg respectively by using a dialysis method, and finally the renatured soluble human interleukin-5 recombinant protein is obtained.
5. SDS-PAGE and protein concentration determination of human interleukin-5 recombinant protein
Mixing 20 μl of above ultrasonic supernatant, post-column liquid, cleaning liquid, and eluent with 20 μl of ACE 2×loading buffer, and boiling in metal bath at 100deg.C for 10min; after centrifugation at 10000rpm for 5min, 10. Mu.L of the supernatant was subjected to SDS-PAGE with a 12% ACE gradient gel, run at 120V for 1h, the results are shown in the right panel of FIG. 1, wherein lane 1 is the pre-purification sample, lane 2 is the post-column pool, lane 3 is the wash pool, lane 4 is the elution pool, and lane 5 is the protein Marker.
It can be seen that the purified expression product has a distinct band at 10kDa, consistent with the predicted molecular weight of human interleukin-5 monomer (10 kDa), and that the purity of the protein of interest is >95%.
The renaturated and concentrated human interleukin-5 is adopted by Thermo Pierce TM BCA Protein Assay Kit the concentration of the purified protein is measured, the content of the target protein is calculated, and finally 100mL of cell expression quantity is obtained, so that the human interleukin-5 recombinant protein with high purity of approximately 1.3mg can be obtained.
6. Activity analysis of human interleukin-5 recombinant protein
Taking renaturated and concentrated human interleukin-5 recombinant protein, and carrying out activity detection by using a Siro biological immunofluorescence cytokine combined detection kit (registration certificate number: ganmechanical injection 20192400359), wherein the detection mode is a double-antibody sandwich method, and the content of the active human interleukin-5 protein is detected.
And (3) diluting the purified human interleukin-5 recombinant protein to 10ng/mL, 5ng/mL, 2.5ng/mL and 1ng/mL respectively by adopting a standard dilution in a cytokine joint detection kit, and then carrying out activity detection by using a flow fluorescence method. The result shows that the fluorescence value of the human interleukin-5 recombinant protein is 123053.60 when the human interleukin-5 recombinant protein is diluted to 2.5ng/mL, and the fluorescence value of the standard substance is 75231.60 when the human interleukin-5 recombinant protein is diluted to 2.5ng/mL, so that the human interleukin-5 recombinant protein has higher activity compared with the human interleukin-5 protein standard substance.
Example 2: purity and bioactivity verification of human interleukin-5 recombinant protein
In this example, a nucleic acid molecule (optimized sequence) shown in SEQ ID No. 5 and a nucleic acid molecule (sequence obtained by removing the signal peptide sequence from the original mRNA sequence SEQ ID No. 7) shown in SEQ ID No. 8 were used, and purification and renaturation were performed after passing through an E.coli expression system according to the method provided in example 1, and SDS-PAGE, protein concentration measurement and bioactivity analysis were performed, respectively.
1. SDS-PAGE and protein yield determination
Mixing 20 μl of above ultrasonic supernatant, post-column liquid, cleaning liquid, and eluent with 20 μl of ACE 2×loading buffer, and boiling in metal bath at 100deg.C for 10min; after centrifugation at 10000rpm for 5min, 10. Mu.L of the supernatant was subjected to SDS-PAGE with a 12% ACE gradient gel, run at 120V for 1h, and the results are shown in FIG. 1, wherein the left panel shows the SDS-PAGE result of human interleukin-5 recombinant protein prepared from the original sequence (SEQ ID NO: 8), the right panel shows the SDS-PAGE result of human interleukin-5 recombinant protein prepared from the optimized sequence (SEQ ID NO: 5), lane 1 shows the sample before purification, lane 2 shows the post-column harvest, lane 3 shows the wash harvest, lane 4 shows the elution harvest, and lane 5 shows the protein Marker.
As can be seen from FIG. 1, the two nucleic acid molecule expression products have obvious bands at 13kDa and 10kDa, respectively, which are consistent with the predicted molecular weight of human interleukin-5 monomer, and can successfully prepare human interleukin-5 recombinant protein.
Two groups of human interleukin-5 recombinant proteins were purified and renatured according to example 1, and the renatured concentrated human interleukin-5 was purified using Thermo Pierce TM BCA Protein Assay Kit the concentration of the purified protein, the volume of bound protein solution, and the final yield measurements are shown in Table 1.
TABLE 1 production of two groups of human interleukin-5 recombinant proteins
| Nucleic acid molecules | Yield (mg/100 mL bacterial liquid expression) |
| SEQ ID NO. 5 (optimized sequence) | 1.3 |
| SEQ ID NO. 8 (original sequence) | 0.704 |
Therefore, the human interleukin-5 recombinant protein prepared by the nucleic acid molecule (SEQ ID NO: 5) provided by the invention can be prepared into target protein (> 95%) with higher protein yield and higher purity by only one-step purification.
2. Analysis of biological Activity of human Interleukin-5 recombinant protein
Two groups of renaturated and concentrated human interleukin-5 recombinant proteins are respectively taken, standard diluent in a cytokine joint detection kit is respectively diluted to 10ng/mL, 5ng/mL, 2.5ng/mL and 1ng/mL, and then an Sier biological immunofluorescence cytokine joint detection kit (registration certificate number: ganzhen injection 20192400359) is used for activity detection on a Canto II flow cytometer of BD company, and the detection results are shown in Table 2.
TABLE 2 Activity of two groups of human interleukin-5 recombinant proteins
As can be seen from Table 2, the human interleukin-5 recombinant protein prepared by the nucleic acid molecule (SEQ ID NO: 5) provided by the invention obviously has higher activity.
Example 3: screening of epitope sequences
In the construction process of the human interleukin-5 recombinant protein coding gene, the method predicts and screens the epitope of the human interleukin-5 based on parameters such as hydrophobicity, protein secondary structure, epitope and the like, and specifically comprises the following steps:
1. protein hydrophobicity prediction
By passing throughhttps://www.expasy.org/resources/protscaleThe amino acid sequence of human interleukin-5 was predicted for protein hydrophobicity, and the predicted results are shown in FIG. 2.
As can be seen from FIG. 2, the prediction selects Ile20-Ser134 sequences (i.e. having amino acids 1-115) other than the signal peptide, and fragments with strong hydrophilicity (predicted value less than 0) are selected as possible epitopes in the prediction results, i.e. 57-63, 75-85, 100-110.
2. Protein secondary structure
By passing throughhttps://www.uniprot.org/uniprotkb/P05113/entryThe amino acid sequence of human interleukin-5 is predicted to have a protein secondary structure, and the detection result is shown in figure 3.
According to FIG. 3, the helix (mauve) and folding strand (yellow) regions are the more difficult epitope-forming regions, and the green is two disulfide-forming regions. The non-helical and folding regions and the sequences comprising disulfide-bond forming regions, i.e., 45-50, 63, 75-82, 103, are selected, possibly epitope sequences.
3. Antigenic determinants
By passing throughhttp://www.detaibio.com/tools/index.phpr＝epitope-prediction％ 2FindexThe amino acid sequence of human interleukin-5 was predicted for an epitope, and the predicted results are shown in FIG. 4.
According to FIG. 5, the prediction selects the Ile20-Ser134 sequence (i.e., having amino acids 1-115) in addition to the signal peptide, and selects the regions with higher scores in the epitope analysis results, i.e., 57-63, 72-84, 98-113, 124-128, possibly as epitope sequences.
Combining the analysis, selecting three sequences which can form epitope according to the predicted results of hydrophobicity, protein secondary structure and antigenic determinant, namely Ala45-Cys63, ser75-Thr82 and Lys103-Arg 110; considering that IL-5 bioactivity may be related to its C-terminal residue and dimer structure, C-terminal Val124-Ser134 was chosen as the fourth sequence.
By comparison, ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 respectively have nucleotide sequences shown in SEQ ID NO 1-4.
In this example, the preparation of nucleic acid molecules was attempted by selecting different epitope sequences, repeating the expression of the epitope sequences, purifying and renaturating the prepared nucleic acid molecules by using an E.coli expression system, and performing SDS-PAGE, protein concentration measurement and biological activity analysis, respectively, thereby screening the nucleic acid molecules for preparing human interleukin-5 recombinant proteins having higher activity, higher purity and easier purification.
5 groups of nucleic acid molecules are selected to express human interleukin-5 recombinant protein as follows:
1. selecting 4 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) of possibly forming antigen epitope sequences for continuous expression, connecting the sequences by using a flexible connecting peptide sequence GGTGGTGGTGGTAGCGGTGGTGGTGGTAGC, and repeatedly expressing the sequences twice, wherein the nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO. 5;
2. selecting 4 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) of sequences which are likely to form an epitope for continuous expression, connecting the sequences by using a rigid connecting peptide sequence GAAGCTGCGGCAAAAGAAGCAGCGGCTAAA, and repeatedly expressing the sequences twice, wherein the nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO. 9; the expressed rigid connecting peptide has an amino acid sequence shown as SEQ ID NO. 10.
3. The sequence of the possible antigen epitope formed by 4 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) is selected for continuous expression, repeated expression is not performed, and the nucleotide sequence of the nucleic acid molecule is as follows:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGT GTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATG AACACCGAGTGGATTATCGAGAGCTGA(SEQ ID NO:11)
4. the sequence of the possible epitope formed by 4 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg110, val124-Ser 134) is selected for continuous expression, the connection is not formed in the middle, the expression is repeated twice, and the nucleotide sequence of the nucleic acid molecule is as follows: ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCTGA (SEQ ID NO: 12)
5. The sequence of 3 segments (Ala 45-Cys63, ser75-Thr82, lys103-Arg 110) which possibly forms an epitope is selected for continuous expression, and is connected by using a flexible connecting peptide sequence GGTGGTGGTGGTAGCGGTGGTGGTGGTAGC in the middle, and the expression is repeated twice, wherein the nucleotide sequence of the nucleic acid molecule is as follows:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCTGA(SEQ ID NO:13)
6. the Ile20-Ser134 sequence (SEQ ID NO: 8) truncated from the original sequence (sequence of the original mRNA sequence SEQ ID NO:7 with the signal peptide sequence removed)
Human interleukin-5 recombinant proteins were prepared according to the method provided in example 1 using 6 sets of nucleic acid molecules, respectively, and their yields and activities were examined, and the results are shown in table 3.
Table 3, 6 production and Activity of human interleukin-5 recombinant proteins
The concentration and purity of the human interleukin-5 recombinant protein prepared by the nucleic acid molecule (SEQ ID NO: 5) provided by the invention can reach higher level, more importantly, the recombinant protein has very high activity, and the human interleukin-5 recombinant protein with higher activity than the human interleukin-5 protein standard can be prepared by only one-step purification, so that the recombinant protein can be used for preparing the standard and also can be used as an antigen for preparing antibodies of immune animals. Comparison of the data from groups 1 and 2 shows that the connecting peptide also affects the activity of the recombinant protein, wherein the flexible connecting peptide of the first group of SEQ ID NO. 5 nucleic acid molecules is more beneficial to the recombinant protein than the second group of rigid connecting peptide SEQ ID NO. 10, indicating that the flexible connecting peptide is more beneficial to the protein folding to form a stable secondary structure.
Example 4: optimization of renaturation process
In this example, a recombinant human interleukin-5 protein was prepared according to the method provided in example 1, wherein in the purification and renaturation process, different buffer systems such as 50mM MOPS, HEPES, PBS, TRIS shown in table 4 were respectively used to prepare renaturation solutions, other components in the renaturation solutions remained unchanged, and the protein yield and protein reactivity after renaturation were respectively detected, and the detection method of the protein yield was as follows: the protein yield was measured by the method provided in example 1 to calculate the total protein amount obtained finally, and the ratio of the total protein amount to the amount of protein bacteria used was the protein yield.
The detection method of protein reactivity comprises the following steps: the detection of protein activity was performed using the method provided in example 2, and the fluorescence value measured per ng of the target protein/the fluorescence value of the protein measured per ng of the standard was calculated.
The influence of renaturation solution prepared by different buffer systems on renaturation effect of human interleukin-5 recombinant protein is examined, and the detection results are shown in Table 4.
TABLE 4 influence of different renaturation solutions on the preparation of human interleukin-5 recombinant protein
| pH value of | Buffer system | Protein yield/50 mL bacterial liquid | Protein reactivity (ng/ng) |
| 6 | MOPS | 530μg±50μg | 1.2 |
| 7 | HEPES | 500μg±50μg | 1.9 |
| 7.4 | PBS | 560±50μg | 1.3 |
| 8 | TRIS | 610±50μg | 2.2 |
| 9 | TRIS | 650±50μg | 1.3 |
As can be seen from Table 4, the recovery solution prepared by using the Tris buffer system with pH9.0 has the highest yield of human interleukin-5 recombinant protein, but has lower protein reactivity (activity), and the recovery solution prepared by using the Tris buffer system with pH8.0 can remarkably improve the protein reactivity, and the yield is slightly reduced compared with that of the Tris buffer system with pH9.0, but still has higher level, so that the preferred recovery solution has the formula: 50mM Tris, 0.5M NaCl, 0.3M Arg, 2mM/1mM GSH/GSSG and 1mM DTT, pH8.0.
Example 5: optimization of purification process
In this example, human interleukin-5 recombinant protein was prepared according to the method provided in example 1, wherein in the purification process, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 70mM, 80mM, 90mM, 100mM imidazole concentrations as shown in Table 5 were used to prepare denatured wash solutions, the other components in the denatured wash solutions were kept unchanged, and the loss rate of the target protein in the wash solution and the total protein proportion of the target protein in the eluate were measured, respectively.
The detection method of the target protein loss rate comprises the following steps: target band gray value in wash solution 2.5/(target band gray value in wash solution 2.5+ target band gray value in eluent).
The detection method for the proportion of the target protein to the total protein of the eluent comprises the following steps: target band gray value in eluent/gray value of all protein bands in eluent.
The effect of using denatured wash solutions of different imidazole concentrations on the preparation of human interleukin-5 recombinant protein was examined and the detection results are shown in table 5.
TABLE 5 influence of denatured wash solutions using different imidazole concentrations on the preparation of human interleukin-5 recombinant protein
| Imidazole concentration | Loss rate of target protein | The target protein accounts for the total protein proportion of the eluent |
| 5mM | 7％ | 76％ |
| 10mM | 10％ | 77％ |
| 20mM | 15％ | 75％ |
| 30mM | 18％ | 80％ |
| 40mM | 20％ | 82％ |
| 50mM | 25％ | 85％ |
| 60mM | 35％ | 86％ |
| 70mM | 40％ | 89％ |
| 80mM | 42％ | 96％ |
| 90mM | 65％ | 97％ |
| 100mM | 75％ | 97％ |
As can be seen from Table 5, the use of the denatured washing liquid at 80mM imidazole concentration can remove impurities to a greater extent and reduce the loss of the target protein while maintaining the protein purity at a higher level, and the purification can be achieved in one step, and the purity of the purified protein can reach more than 95%.
Whereas the human interleukin-5 recombinant Protein prepared in the prior art generally requires multiple purification steps (e.g., FEBS Lett. (1993) 27;331 (1-2): 49-52.Doi:10.1016/0014-5793 (93) 80295-6./Protein Expr Purif. (1997) 11 (1): 86-94.Doi: 10.1006/prep.1997.0785.) because the purity of the target Protein (68%) obtained by one-step column purification is significantly lower, it is difficult to prepare high-purity human interleukin-5 recombinant Protein by one-step purification.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Sequence listing
Seq ID NO.1
Ala45-Cys63：
GCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGT
Seq ID NO.2
Ser75-Thr82：
AGCCAAACCGTTCAGGGCGGTACG
Seq ID NO.3
Lys103-Arg110：
AAGTGCGGCGAGGAGCGCCGTCGC
Seq ID NO.4
Val124-Ser134：
GTTATGAACACCGAGTGGATTATCGAGAGC
SEQ ID NO:5
Nucleic acid molecule encoding a human interleukin-5 recombinant protein:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCTGA
SEQ ID NO:6
recombinant protein sequence of human interleukin-5:
ANETLRIPVPVHKNHQLCSQTVQGGTKCGEERRRVMNTEWIIESGGGGSGGGGSANETLRIPVPVHKNHQLCSQTVQGGTKCGEERRRVMNTEWIIES
SEQ ID NO:7
mRNA sequence of human interleukin-5:
ATGCACTTTCTTTGCCAAAGGCAAACGCAGAACGTTTCAGAGCCATGAGGATGCTTCTGCATTTGAGTTTGCTAGCTCTTGGAGCTGCCTACGTGTATGCCATCCCCACAGAAATTCCCACAAGTGCATTGGTGAAAGAGACCTTGGCACTGCTTTCTACTCATCGAACTCTGCTGATAGCCAATGAGACTCTGAGGATTCCTGTTCCTGTACATAAAAATCACCAACTGTGCACTGAAGAAATCTTTCAGGGAATAGGCACACTGGAGAGTCAAACTGTGCAAGGGGGTACTGTGGAAAGACTATTCAAAAACTTGTCCTTAATAAAGAAATACATTGACGGCCAAAAAAAAAAGTGTGGAGAAGAAAGACGGAGAGTAAACCAATTCCTAGACTACCTGCAAGAGTTTCTTGGTGTAATGAACACCGAGTGGATAATAGAAAGTTGAGACTAAACTGGTTTGTTGCAGCCAAAGATTTTGGAGGAGAAGGACATTTTACTGCAGTGAGAATGAGGGCCAAGAAAGAGTCAGGCCTTAATTTTCAGTATAATTTAACTTCAGAGGGAAAGTAAATATTTCAGGCATACTGACACTTTGCCAGAAAGCATAAAATTCTTAAAATATATTTCAGATATCAGAATCATTGAAGTATTTTCCTCCAGGCAAAATTGATATACTTTTTTCTTATTTAACTTAACATTCTGTAAAATGTCTGTTAACTTAATAGTATTTATGAAATGGTTAAGAATTTGGTAAATTAGTATTTATTTAATGTTATGTTGTGTTCTAATAAAACAAAAATAGACAACTGTT
SEQ ID NO:8
Ile20-Ser134：
ATGATCCCCACAGAAATTCCCACAAGTGCATTGGTGAAAGAGACCTTGGCACTGCTTTCTACTCATCGAACTCTGCTGATAGCCAATGAGACTCTGAGGATTCCTGTTCCTGTACATAAAAATCACCAACTGTGCACTGAAGAAATCTTTCAGGGAATAGGCACACTGGAGAGTCAAACTGTGCAAGGGGGTACTGTGGAAAGACTATTCAAAAACTTGTCCTTAATAAAGAAATACATTGACGGCCAAAAAAAAAAGTGTGGAGAAGAAAGACGGAGAGTAAACCAATTCCTAGACTACCTGCAAGAGTTTCTTGGTGTAATGAACACCGAGTGGATAATAGAAAGTTGA
SEQ ID NO:9
altering the recombinant protein molecule nucleic acid sequence of the connecting peptide:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCGAAGCTGCGGCAAAAGAAGCAGCGGCTAAAGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCTAA
SEQ ID NO:10
altering the amino acid sequence of the recombinant protein molecule linking peptide of the linking peptide:
EAAAKEAAAK
SEQ ID NO:11
group 3 nucleic acid molecules:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCTGA
SEQ ID NO:12
group 4 nucleic acid molecules:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGTTATGAACACCGAGTGGATTATCGAGAGCTGA
SEQ ID NO:13
group 5 nucleic acid molecules:
ATGGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGCTAATGAAACCTTACGTATTCCGGTTCCGGTGCACAAGAACCACCAACTGTGTAGCCAAACCGTTCAGGGCGGTACGAAGTGCGGCGAGGAGCGCCGTCGCTGA
Claims (10)
1. a nucleic acid molecule for encoding a human interleukin-5 recombinant protein, comprising two repeated Ala45-Cys63, ser75-Thr82, lys103-Arg110, and Val124-Ser134 sequences, said Ala45-Cys63, ser75-Thr82, lys103-Arg110, and Val124-Ser134 having the nucleotide sequences shown in SEQ ID NOs 1-4, respectively.
2. The nucleic acid molecule of claim 1, having a nucleotide sequence set forth in SEQ ID NO. 5.
3. A human interleukin-5 recombinant protein encoded by the nucleic acid molecule of claim 1 or 2.
4. The recombinant human interleukin-5 protein of claim 3, having an amino acid sequence as set forth in SEQ ID NO. 6.
5. A method for preparing human interleukin-5 recombinant protein, characterized in that a recombinant vector comprising the nucleic acid molecule of claim 1 or 2 is constructed and expressed by a prokaryotic system.
6. The method of manufacturing as claimed in claim 5, comprising the steps of:
(1) Inserting the nucleic acid molecule into an escherichia coli expression vector to obtain a recombinant vector;
(2) The recombinant vector is transformed into an escherichia coli expression system to obtain a bacterial solution;
(3) Adding denatured lysate into the thallus solution for cracking and centrifuging to obtain protein solution;
(4) Purifying the protein solution by a column, and washing and eluting to obtain protein eluent;
(5) Adding renaturation solution into the protein eluent, ultrafiltering, concentrating and dialyzing to obtain the human interleukin-5 recombinant protein.
7. The method of claim 6, wherein the denaturing wash in step (4) has a pH of 8.0 and comprises 0.01M PBS,8M urea, and 80mM imidazole.
8. The method of claim 6, wherein the renaturation solution in step (5) contains Tris at a pH of 8.0.
9. The method of claim 6, wherein the renaturation solution of step (5) comprises 50mM Tris, 0.5M NaCl, 0.3M Arg, 2mM/1mM GSH/GSSG and 1mM DTT, pH8.0.
10. Use of a nucleic acid molecule comprising the sequence Ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 repeated twice for the preparation of a highly active human interleukin-5 recombinant protein, wherein said Ala45-Cys63, ser75-Thr82, lys103-Arg110 and Val124-Ser134 have the nucleotide sequences as shown in SEQ ID NOs 1 to 4, respectively.
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| US5681936A (en) * | 1994-03-17 | 1997-10-28 | Merck Frosst Canada, Inc. | Method of purification of recombinant human interleukin-5 |
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| US20040076611A1 (en) * | 2002-07-17 | 2004-04-22 | Bachmann Martin F. | Molecular antigen arrays |
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| US5681936A (en) * | 1994-03-17 | 1997-10-28 | Merck Frosst Canada, Inc. | Method of purification of recombinant human interleukin-5 |
| US20020127200A1 (en) * | 1998-05-29 | 2002-09-12 | Shumin Yang | Canine and feline immunoregulatory proteins, nucleic acid molecules, and uses thereof |
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