Preparation method and application of functional active body of Chemerin
Through the secretion and expression in insect cells and purification of multi-layer ultrafiltration membranes, the problem of incorrect folding of baogalactin functional actives in prokaryotes was solved, and a functional actives in baogalactin with complete physiological functions was obtained. In inflammation and fat regulation.
Patent Information
- Application Number
- CN202411686108.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-24
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art is difficult to correctly fold and express chemotamin functional activators in prokaryotes such as E. coli, resulting in physiological functional defects in their interactions with chemotamin receptors.
By using the method of secreting and expressing recombinant human chemotamin functional actives, basal infection insect cells were introduced through homologous recombination, impurity ions were removed by precipitation of calcium chloride, and purification was carried out through multi-layer ultrafiltration membrane to finally obtain complete Chemotamin functional activator for physiological functions.
The correct folding and efficient purification of the functional active body of baogalin is achieved, ensuring that it can form a two-site binding mode with the baogalin receptor, and has complete physiological functions, providing a basis for the study of inflammation and fat regulation.
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Figure CN119432916A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a recombinant chemokine functional active body. Background Art
[0002] Chemokines are important atypical chemokines and adipokines, also known as retinoic acid receptor responsive protein 2 and tazarotene-induced protein 2. Chemokines exert their biological functions through two G protein-coupled receptors, chemokine receptor 1 and chemokine receptor 2. Chemokines and chemokine receptors are expressed in immune tissues and white adipose tissue. In recent years, they have been found to play an important role in leukocyte chemotaxis, adipocyte differentiation and lipolysis, and can regulate the occurrence and development of inflammation and obesity.
[0003] Chemotactin needs to undergo complex enzymatic cleavage and processing to form a functionally active body with physiological functions when expressed and secreted under physiological conditions. Chemotactin is first expressed and translated into a chemokine protomer, which contains 163 amino acids. During the secretion maturation process, the signal peptide of 20 amino acids at the N-terminus is removed. In addition, some amino acids need to be hydrolyzed at the C-terminus to form a functionally active body. Among them, the form with the highest functional activity is a 157-amino acid functionally active body containing amino acids 21-157 of the protomer after the removal of 6 amino acids at the C-terminus. The functionally active body of chemokine contains 3 pairs of disulfide bonds, and more importantly, the special processing and folding process makes it difficult to fold correctly in prokaryotes such as Escherichia coli. Chemotactin is recombinantly expressed in Escherichia coli in the form of inclusion bodies. After inclusion body refolding and purification, the biological function is close to that of the artificially synthesized C-terminal 9 peptide, indicating that its N-terminal domain is not correctly folded. Further studies have shown that the chemokine purified by inclusion body refolding cannot form a two-site binding mode with the chemokine receptor, indicating that the N-terminal domain of chemokine is difficult to spontaneously complete the correct assembly and folding.
[0004] In our study, we found that the interaction between chemokine and chemokine receptor is divided into two regions, CBR1 and CBR2. The C-terminal 9 peptide of artificially synthesized chemokine only interacts with CBR2 and has obvious physiological function defects. The functional active body of chemokine with correctly folded N-terminal domain has complete physiological function. Summary of the invention
[0005] The present invention provides a method for preparing a functional active form of chemokine by recombinant expression and purification, and provides a protein preparation method for further functional research and anti-inflammatory and lipid-lowering applications of chemokine. The amino acid sequence of the functional active form of chemokine prepared by the present invention is shown in SEQ ID NO.1.
[0006] The present invention provides the following technical solutions:
[0007] The present invention provides a preferred recombinant human chemokine protein nucleotide sequence, as shown in SEQ ID NO.2.
[0008] The present invention provides a method for secreting and expressing a recombinant human chemokine functionally active body through insect cells, comprising the following steps:
[0009] (1) The nucleotide sequence SEQ ID NO. 2 is introduced into the bacmid by homologous recombination and then infected into insect cells.
[0010] (2) Remove cells and debris by centrifugation and harvest the clarified solution containing the target protein.
[0011] Preferably, the insect cells in step (1) are sf9 insect cells.
[0012] Preferably, the infection ratio MOI in step (1) is 1.
[0013] Preferably, the culture product is collected in step (2) 96 hours after the addition of the bacmid.
[0014] Preferably, the centrifugal acceleration in step (2) is 2000g-4000g to reduce the release of cell contents.
[0015] The present invention provides a method for purifying the functionally active form of chemokine from the expression product, comprising the following steps:
[0016] (1) Adding calcium chloride introduces chloride ions and calcium ions and precipitates impurity ions dissolved in the culture medium.
[0017] (2) Add Tris base to adjust the solution pH to 7.4 and stabilize the pH environment.
[0018] (3) Remove insoluble precipitates by centrifugation and further remove tiny precipitates by passing through a microporous filter membrane.
[0019] (4) Use large-pore ultrafiltration membrane to remove large molecular weight protein impurities.
[0020] (5) The target protein chemokine is concentrated through a small-pore ultrafiltration membrane, and the solution components are replaced with physiological saline and buffer salt.
[0021] Preferably, the centrifugal speed in step (3) is 4500 rpm and the centrifugal time is 30 minutes.
[0022] Preferably, the pore size of the microporous filter membrane in step (3) is 0.2 to 0.45 microns.
[0023] Preferably, the ultrafiltration membrane cutoff size in step (4) is 30 kD to 1000 kD, or the pore size is 5 nm to 100 nm.
[0024] Preferably, the ultrafiltration membrane cut-off size in step (5) is 3 kD.
[0025] The present invention also provides a use of the recombinant human chemokine functionally active body in the research and development of inflammation and fat regulation.
[0026] Beneficial effects of the present invention:
[0027] (1) The recombinant expression method provided by the present invention adopts secretory expression. The recombinant protein is secreted into a large volume of culture medium, which has a higher yield than small volume expression in cells or bacteria.
[0028] (2) The recombinant expression method provided by the present invention is based on the inventor's research findings that insect cells can effectively recognize and cleave the signal peptide amino acid sequence MRRLLIPLALWLGAVGVGVA. By designing a technical solution to cleave the signal peptide during the insect cell secretion process, the technical bottleneck of recombinant expression of the chemokine functional active body with glutamic acid as the first amino acid instead of methionine corresponding to the start codon is overcome. At present, the recombinant expression of most proteins requires the first amino acid to be designed as methionine corresponding to the start codon.
[0029] (3) The recombinant expression method provided by the present invention uses inoculation of bacmids for expression. Bacmids can replicate themselves and be recycled and reused. Compared with the cumbersome large-scale plasmid extraction required for mammalian cell recombinant expression, the preparation method of the present invention only requires two small-scale DNA extractions.
[0030] (4) The preparation method provided by the present invention has simple steps and does not require column chromatography steps such as affinity column chromatography and gel exclusion column chromatography, and in vitro enzymatic cleavage of protein tags, which are bottleneck steps that increase costs and limit yields. The convenient operation process improves efficiency and reduces costs, laying a solid foundation for the application of the present invention.
[0031] (5) The preparation method provided by the present invention does not introduce any extra amino acids. Conventional recombinant expression methods require the fusion of tag sequences for purification, which affect the folding and function of chemokines. In addition, completely removing the tags without leaving any extra amino acids is also a high-cost and rate-limiting step in the field of protein engineering.
[0032] (6) The preparation process provided by the present invention does not introduce endotoxins. It is difficult to completely remove endotoxins through conventional methods of bacterial recombinant expression and purification under existing technologies, and bacterial endotoxins themselves have immune effects and can cause inflammation. Low levels of endotoxins can also interfere with experimental results of immune-inflammation related studies.
[0033] (7) The chemokine prepared by the present invention has been shown to be able to bind to all known functional receptors of chemokine, namely chemokine receptor 1 and chemokine receptor 2, and form a dual-site binding pattern, which provides a basis for the application of the chemokine functional active body prepared by the method of the present invention in inflammation and fat regulation. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In order to more clearly illustrate the technical solutions in the specific implementation manner of the present invention, the drawings required for describing the specific implementation manner are briefly introduced below.
[0035] Figure 1 It is a schematic diagram of the technical solution for constructing the chemokine nucleotide sequence into pFastbac1;
[0036] Figure 2 It is a schematic diagram of the technical principle of secretory expression;
[0037] Figure 3 It is a schematic diagram of the technical route of extraction and purification;
[0038] Figure 4 This is a diagram showing the results of SDS-PAGE gel electrophoresis of the purified chemokine functional active body;
[0039] Figure 5 This is a graph showing the results of biological functional activity testing of the purified chemokine functionally active body;
[0040] Figure 6 This is a functional test result diagram of the dual-site activity of the purified chemokine functional active body;
[0041] Figure 7 This is the molecular structure diagram of the purified chemokine functional active body;
[0042] Figure 8 This is a molecular structure diagram of the purified chemokine functional active body that activates the chemokine receptor 1 at two sites;
[0043] Fig. 9 This is a molecular structure diagram of the purified chemokine functional active body that activates chemokine receptor 2 at two sites. DETAILED DESCRIPTION
[0044] In order to more clearly describe the technical solution of the present invention, it is further described below in conjunction with specific embodiments, but it does not constitute a limitation on the embodiments of the present invention.
[0045] The embodiment provided by the present invention comprises the following steps:
[0046] 1. Molecular construction of recombinant human chemokine.
[0047] The molecular construction and operation methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and reference books and reference books such as Molecular Cloning Experiment Guide published by Science Press can be referred to. The reagents and consumables used are commercially available products unless otherwise specified.
[0048] (1) Based on the amino acid sequence of the chemokine functional active body, as shown in SEQ ID NO.1, a preferred nucleotide sequence as shown in SEQ ID NO.2 is obtained by double-stranded DNA chemical synthesis method, amplified by PCR and seamlessly connected to the polyhedrin promoter of pFastbac1 vector by homologous recombination, as shown in Figure 1 shown.
[0049] (2) The homologous recombination product was transformed into DH5alpha competent cells, spread on LB plates containing ampicillin antibiotics, and cultured overnight.
[0050] (3) Select 10 single colonies, inoculate them into 5 mL of LB liquid medium containing ampicillin, and culture overnight.
[0051] (4) Perform plasmid extraction on 10 samples of overnight culture.
[0052] (5) Use universal primers to perform PCR amplification verification, detect the fragment size of the PCR product by agarose gel electrophoresis, and screen one positive construct.
[0053] 2. Preparation of bacmid.
[0054] (1) Transform the selected positive construct into DH10bac competent bacteria with the plasmid, spread it on a plate containing 0.05 mg / mL kanamycin, 0.01 mg / mL tetracycline, 0.007 mg / mL gentamicin, 0.1 mg / mL X-gal, and 0.04 mg / mL IPTG, and culture overnight at 37°C.
[0055] (2) Select 5 white colonies and inoculate them into 5 mL of LB liquid medium containing 0.05 mg / mL kanamycin, 0.01 mg / mL tetracycline, and 0.007 mg / mL gentamicin. Incubate the culture overnight at 37 degrees with shaking at 220 rpm.
[0056] (3) Bacmid was extracted by the classic alkaline lysis method, and PCR was performed using universal primers M13F and M13R. The size of the PCR-amplified DNA fragment was verified by agarose gel electrophoresis.
[0057] (4) The verified Bacmid was transfected into Sf9 insect cells using the classic liposome method and the bacmid was harvested.
[0058] 3. Expression of recombinant human chemokine (technical principle see Figure 2 ).
[0059] (1) Culture sf9 insect cells in suspension at 27°C and 110 rpm.
[0060] (2) When the cell density is 2 x 106 cells / mL, the bacmid is inoculated, the MOI is 1, and the culture volume is 1 L.
[0061] (3) Continue culturing at 27 degrees and 110 rpm for 96 hours.
[0062] 4. Purification of recombinant human chemokine (Technical route see Figure 3 )
[0063] (1) Centrifuge at 3000 rpm to remove cells and transfer the supernatant to a clean large beaker. Slowly add 5 mL of 2 M calcium chloride solution, then adjust the pH to 7.4 with 0.1 M sodium hydroxide solution and 0.1 M hydrochloric acid solution, and add 20 mL of 1 M Hepes solution (pH 7.4).
[0064] (2) After stirring at 4 degrees for 15 minutes, let it stand for half an hour, remove the settled precipitate and collect the supernatant.
[0065] (3) Centrifuge at 4500 rpm for 30 minutes. The supernatant after centrifugation is slowly filtered through a 0.45 μm microporous filter at 4 degrees to obtain 800 mL, and then filtered again through a 0.22 μm microporous filter to obtain 700 mL.
[0066] (4) Use a 50 kDa ultrafiltration tube to perform ultrafiltration at a speed of 4500 rpm, with a filtration volume of 500 mL.
[0067] (5) Use a 3 kDa ultrafiltration tube for ultrafiltration at 4000 rpm with a retention volume of 5 mL.
[0068] (6) Add 20 mM Hepes 150 mM sodium chloride solution to a volume of 15 mL and perform ultrafiltration at 4000 rpm with a retention volume of 5 mL.
[0069] (7) Repeat step (6) three times to replace the solution environment.
[0070] (8) The concentration was determined by Nanodrop A280 spectrophotometry and was 1.3 mg / mL.
[0071] 5. Activity verification of recombinant human chemokine
[0072] (1) SDS-PAGE detection shows that the purified protein has a single band, such as Figure 4 shown.
[0073] (2) The functional experimental system of G protein-coupled receptors was used to determine the functional effects of purified chemokines on chemokine receptor 1 and chemokine receptor 2. The results showed that the purified chemokines were biologically active and acted on chemokine receptor 1 and chemokine receptor 2 in a dual-site mode, such as Figure 5-9 shown.
Claims
1. A method for recombinant preparation of a functionally active form of human chemerin protein, characterized in that: The following steps are involved: a. Construct the nucleotide sequence encoding the chemokine into a vector and prepare the recombinant bacmid b. Chemokine secretion expression using insect cells c. Collect the culture medium and purify the chemokine functional active protein by centrifugation and ultrafiltration.
2. The chemokine according to claim 1, characterized in that The amino acid sequence is shown in SEQ ID NO.
1. Preferably, the nucleotide sequence used is shown as SEQ ID No.
2.
3. The preparation method according to claim 1, characterized in that: In step a, the construction is carried out by homologous recombination, and no restriction enzyme cutting sites are involved at both ends of the nucleotide sequence.
4. The preparation method according to claim 1, characterized in that: In step a, the construction method does not involve tag sequences at both ends of the amino acid sequence.
5. The preparation method according to claim 1, characterized in that: In step b, the inoculum MOI value is 1.
6. The preparation method according to claim 1, characterized in that: In step b, the expression time is 96 hours.
7. The preparation method according to claim 1, characterized in that: In step c, the purification of the chemokine functional active body protein comprises the following steps: Cells and cell debris were removed by centrifugation, calcium chloride was added to precipitate impurities, alkaline solution was added to adjust the pH and Hepes was supplemented to stabilize the pH, the precipitate was removed by centrifugation, soluble macromolecular polymers and chemokine proteins were separated by microporous membrane, impurity proteins were retained by ultrafiltration, and proteins were concentrated by ultrafiltration. Preferably, the final concentration of calcium chloride is 10 mM. Preferably, the final concentration of Hepes is 20 mM. Preferably, the pore size of the microporous filter membrane is 0.45 microns. Preferably, the ultrafiltration membrane has a protein cut-off size of 30 kDa. Preferably, the ultrafiltration membrane protein concentration cut-off size is 3 kDa.
8. The preparation method according to claim 1, characterized in that: The prepared chemokine does not contain any superfluous amino acid sequence.
9. The preparation method according to claim 1, characterized in that: The prepared chemokine can form a two-site binding mode with the chemokine receptor.
10. Application of the method for preparing recombinant human chemokine functional active body protein as claimed in claims 1 to 9 in the research and development and industrial production of chemokine anti-inflammatory and lipolysis.