Preparation method and renaturation method of bovine lactoferrin peptide inclusion body

Abstract

The invention provides a preparation method and a renaturation method of a bovine lactoferrin peptide inclusion body, and belongs to the technical field of biology.The preparation method comprises the steps that coding DNA of bovine lactoferrin peptide Bl fFf 344 is designed on the basis of escherichia coli preference codons, then an escherichia coli induced expression vector is constructed on the basis and converted into escherichia coli, and the escherichia coli inclusion body is obtained; genetic engineering recombinant expression escherichia coli is obtained; inducing the genetic engineering recombinant expression escherichia coli to efficiently express the bovine lactoferrin peptide B1 fFf344 by using isopropyl thiogalactoside, and forming an inclusion body, so as to obtain a thallus mixed solution; and centrifuging the thallus mixed solution, collecting escherichia coli thalli, carrying out wall breaking treatment after resuspending, and then centrifugally collecting the bovine lactoferrin inclusion body. According to the method, the bovine lactoferrin peptide inclusion body can be efficiently prepared, and the bovine lactoferrin peptide is efficiently produced through genetic engineering by combining with the established method for separating, dissolving, purifying and renaturating the bovine lactoferrin peptide inclusion body.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a preparation method and a renaturation method of bovine lactoferrin peptide inclusion bodies. Background Art

[0002] Using E. coli to express foreign genes is the main method for producing protein polypeptides by genetic engineering. Since E. coli is easy to ferment and culture and has a relatively complete induction and regulation expression system, it has become the main system for producing foreign proteins by genetic engineering. Compared with other expression systems such as yeast expression system, plant cell system and insect cell system, it has unique advantages of simplicity and high efficiency. It is currently widely used in the genetic engineering production of genetically engineered drugs such as human insulin, human growth hormone, erythropoietin (EPO), etc.

[0003] The biggest challenge in using E. coli to express foreign proteins is that when foreign proteins are synthesized in large quantities in E. coli cells, they are very likely to aggregate into insoluble protein aggregates - inclusion bodies. The formation of inclusion bodies often results in the inability of the target polypeptide to dissolve effectively and show activity, thus rendering production ineffective. However, if effective dissolution and renaturation conditions are established for the inclusion bodies expressed by genetic engineering of a certain protein polypeptide, so that it can dissolve and restore the correct configuration and perform normal functions, the expression of inclusion bodies will be beneficial to the purification of protein polypeptides and improve production efficiency.

[0004] Bovine lactoferrin (LF) is an 80KDa iron-binding glycoprotein that exists in the exocrine fluid (milk) of cattle and certain granules of multinuclear lymphocytes. In addition to its antibacterial activity, it also regulates the body's iron transfer, anti-tumor and anti-virus functions, and can participate in the body's anti-infection and immune processes by regulating a variety of cytokines. Bovine lactoferrin is a new type of antimicrobial peptide that can be used as a promising antibiotic substitute in animal husbandry. Drago-Serrano et al. fed different doses of bovine lactoferrin to rats infected with Salmonella typhimurium and found that bovine lactoferrin could reduce the number of Salmonella typhimurium in rats. The antibacterial activity of bovine lactoferrin is actually exerted by the leaf-like structure of the bovine lactoferrin functional fragment (BlfFf). The two small peptides produced by protease hydrolysis of bovine lactoferrin are located at the N-terminus, namely the 17-41aa bovine lactoferrin peptide (Lfcin B) and the 268-284aa bovine lactoferrin peptide (LF ampin) domains.

[0005] There are many reports and patents on the genetic engineering expression of bovine lactoferrin peptide. Guo Donghua et al. expressed bovine lactoferrin peptide Lfcin B in Escherichia coli. The Lfcin B gene was expressed in large quantities in Escherichia coli. The expressed protein existed in the form of inclusion bodies. The purified Lfcin B recombinant protein inclusion bodies were renatured using TGE dialysis fluid. The antibacterial results showed that the renatured Lfcin B recombinant protein had good antibacterial activity against ampicillin-resistant Escherichia coli. Xing Jun et al. expressed the first 121 amino acids of bovine lactoferrin and obtained an inactive or non-toxic bovine lactoferrin peptide precursor. The yeast expression system has been applied to the expression of bovine lactoferrin. Zha Xiangdong et al. (CN104073511A) carried out the construction of vectors and expression preparation of yeast constitutive expression. Li Bentao et al. (CN103602603A) obtained the patent for the expression and preparation of bovine lactoferrin peptide in Pichia pastoris. Zhang Guanghua et al. (CN117004501B) secreted and expressed bovine lactoferrin antimicrobial peptide in Pichia pastoris. Wang Jianhua et al. (CN102408480A) also disclosed the patent "A yeast-expressed bovine lactoferrin-derived antimicrobial fragment BLfA and its preparation method". The bovine lactoferrin peptide expressed by the prior art often inhibits the growth of the host bacteria, and the purification and preparation efficiency of the protein product is low, and the manufacturing cost is relatively high. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for preparing bovine lactoferrin peptide inclusion bodies, which can efficiently prepare bovine lactoferrin peptide inclusion bodies, and the bovine lactoferrin peptide inclusion bodies have almost no effect on the growth of host bacteria and are easy to purify, and can significantly improve the production efficiency of bovine lactoferrin peptide. Combined with the separation, dissolution, purification and renaturation methods established for bovine lactoferrin peptide inclusion bodies, efficient production of bovine lactoferrin peptide by genetic engineering is achieved.

[0007] The invention also provides a method for renaturing bovine lactoferrin peptide inclusion bodies.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing bovine lactoferrin peptide inclusion bodies, the preparation method comprising:

[0010] Designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codons of Escherichia coli, and constructing an Escherichia coli inducible expression vector with the coding DNA;

[0011] Transforming the E. coli inducible expression vector into E. coli to obtain genetically engineered recombinant expression E. coli;

[0012] Inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344 with isopropylthiogalactoside, and the expression product forms bovine lactoferrin peptide inclusion bodies to obtain a bacterial cell mixture;

[0013] The bacterial mixture is centrifuged to collect the Escherichia coli cells, which are resuspended and then broken, and then centrifuged to collect the bovine lactoferrin peptide inclusion bodies.

[0014] Furthermore, the coding DNA of bovine lactoferrin peptide BlfFf 344 is designed based on the preferred codons of Escherichia coli, and the coding DNA is used to construct an Escherichia coli inducible expression vector, which specifically includes:

[0015] Designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codons of Escherichia coli, and homologously recombining the coding DNA with the linearized vector pET to obtain an Escherichia coli inducible expression vector;

[0016] Wherein, the amino acid sequence of the bovine lactoferrin peptide BlfFf 344 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding DNA is shown in SEQ ID NO.2;

[0017] The E. coli inducible expression vector map is as follows Figure 1 shown.

[0018] Furthermore, the step of transforming the E. coli inducible expression vector into E. coli to obtain genetically engineered recombinant expression E. coli specifically includes:

[0019] The E. coli inducible expression vector is transformed into E. coli BL21 to obtain genetically engineered recombinant expression E. coli.

[0020] Furthermore, the method of inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344 with isopropylthiogalactoside, and the expression product forms bovine lactoferrin peptide inclusion bodies to obtain a bacterial cell mixture, specifically includes:

[0021] The genetically engineered recombinant expression Escherichia coli is inoculated into a liquid culture medium. When the genetically engineered recombinant expression Escherichia coli grows to OD600=0.6±0.1, the culture temperature is controlled to be 25±1°C, isopropylthiogalactoside is added at a final concentration of 1±0.1 mmol / L to induce the efficient expression of the bovine lactoferrin peptide BlfFf 344, and the expression product forms bovine lactoferrin peptide inclusion bodies to obtain a bacterial mixed solution.

[0022] Furthermore, the bacterial mixture is centrifuged to collect the Escherichia coli cells, which are resuspended and then subjected to cell wall breaking treatment, followed by centrifugation to collect the bovine lactoferrin peptide inclusion bodies, which specifically includes:

[0023] Centrifuging the bacterial cell mixture, removing the supernatant, and collecting the Escherichia coli cells;

[0024] The Escherichia coli cells are resuspended and then broken by ultrasonic wave to obtain inclusion body mixed solution;

[0025] The inclusion body mixture is centrifuged, the supernatant is removed, and the precipitate is collected to obtain bovine lactoferrin peptide inclusion bodies.

[0026] Based on the same inventive concept, the present invention provides a method for renaturing bovine lactoferrin peptide inclusion bodies, the method comprising:

[0027] dissolving bovine lactoferrin peptide inclusion bodies in a urea solution to obtain a mixed solution;

[0028] The mixed solution is purified by gel filtration chromatography to obtain a bovine lactoferrin peptide solution;

[0029] adding a refolding solution to the bovine lactoferrin peptide solution and performing dialysis to obtain a bovine lactoferrin peptide refolding solution;

[0030] The bovine lactoferrin peptide renaturation solution was freeze-dried to obtain bovine lactoferrin peptide BlfFf 344.

[0031] Wherein, the bovine lactoferrin peptide inclusion body is prepared by the above-mentioned method for preparing bovine lactoferrin peptide inclusion body.

[0032] Furthermore, the step of dissolving the bovine lactoferrin peptide inclusion bodies in a urea solution to obtain a mixed solution specifically comprises:

[0033] The bovine lactoferrin peptide inclusion bodies were washed and added to 8.0 mol / L urea solution, stirred and dissolved at 4±1°C for 2.0 h, and then ultrasonically assisted melting was performed until the solution became transparent to obtain a mixed solution;

[0034] Wherein, the ratio of the mass of the bovine lactoferrin peptide inclusion body to the volume of the urea solution is 1 g:10 mL.

[0035] Furthermore, the mixed solution is purified by gel filtration chromatography to obtain a bovine lactoferrin peptide solution, which specifically comprises:

[0036] The mixed solution was centrifuged, the supernatant was collected and filtered through a 0.45 μm filter membrane, and then purified through a SuperTandex75prep grade gel column to obtain a bovine lactoferrin peptide solution.

[0037] Furthermore, the step of adding a refolding solution to the bovine lactoferrin peptide solution and then dialyzing to obtain a bovine lactoferrin peptide refolding solution specifically comprises:

[0038] Adding the refolding liquid to the bovine lactoferrin peptide solution, and then filling it into a 14KDa dialysis bag for dialysis to obtain a bovine lactoferrin peptide refolding solution;

[0039] The volume ratio of the bovine lactoferrin peptide solution to the refolding solution is 1:100;

[0040] The composition and concentration of the solute in the refolding solution are as follows:

[0041] Tris.Cl: 50mmol / L, EDTA.Na2: 0.5mmol / L, NaCl: 50mmol / L, oxidized glutathione: 0.05mmol / L, glutathione: 0.5mmol / L, glycerol: 5% (weight percentage).

[0042] Furthermore, the step of adding a refolding solution to the bovine lactoferrin peptide solution and then placing the solution in a 14KDa dialysis bag for dialysis to obtain a bovine lactoferrin peptide refolding solution specifically comprises:

[0043] Adding the refolding solution to the bovine lactoferrin peptide solution, placing the solution in a 14KDa dialysis bag for dialysis at a dialysis temperature of 4±1°C, replacing the dialysis solution once every 12 hours, and replacing the dialysis solution three times in total to obtain a bovine lactoferrin peptide refolding solution;

[0044] Wherein, the dialysate is 1XPBS buffer with a pH of 7.4 (NaCl: 137mM, KCl: 2.7mM, Na2HPO4: 10mM, KH2PO4: 2mM).

[0045] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0046] 1. The invention discloses a method for preparing bovine lactoferrin peptide inclusion bodies. The method selects bovine lactoferrin N-terminal 344aa as a target polypeptide, first designs a coding DNA of bovine lactoferrin peptide BlfFf 344 based on the codon preference of an Escherichia coli expression system, constructs an Escherichia coli inducible expression vector with the coding DNA, transforms the expression vector into Escherichia coli, induces efficient expression of the recombinant gene in the engineered Escherichia coli with IPTG (isopropylthiogalactoside), forms insoluble bovine lactoferrin peptide inclusion bodies with the expression product, collects and crushes the Escherichia coli cells, and obtains the bovine lactoferrin peptide inclusion bodies. The method can efficiently express the bovine lactoferrin peptide without affecting the growth of the host bacteria by optimizing the coding DNA codon design and using the Escherichia coli expression system for inclusion body expression. The expression product forms inclusion bodies, which is beneficial to the purification of the bovine lactoferrin peptide BlfFf 344 and improves the efficiency of producing bovine lactoferrin functional peptides by genetic engineering.

[0047] 2. The present invention discloses a method for preparing bovine lactoferrin peptide inclusion bodies. The method expresses bovine lactoferrin functional polypeptide in Escherichia coli by inclusion body expression. The bovine lactoferrin peptide BlfFf 344 contains two functional domains, LF ampin and Lfcin B. Experiments show that the bovine lactoferrin peptide BlfFf344 prepared by the present invention exhibits complete polypeptide activity after dissolution and renaturation of the inclusion bodies, and can release antimicrobial peptides LF ampin and Lfcin B after being treated with pepsin.

[0048] 3. A method for renaturing bovine lactoferrin peptide inclusion bodies of the present invention, wherein the bovine lactoferrin peptide inclusion bodies are dissolved by using urea of ​​a certain concentration under established physical conditions, and the obtained mixed solution is purified by gel filtration chromatography to obtain a single bovine lactoferrin peptide, and a renaturation solution is added during the dialysis purification process, and the purified bovine lactoferrin peptide BlfFf 344 exhibits complete polypeptide activity and can release antimicrobial peptides LF ampin and Lfcin B after being treated with pepsin. Experiments have shown that, in combination with the preparation method and renaturation method of the bovine lactoferrin peptide inclusion bodies of the present invention, 1L of culture solution containing genetically engineered recombinant expression Escherichia coli (wet weight of bacteria 5-8g, dry weight 0.5-0.8g) can produce 100-150mg of active bovine lactoferrin peptide BlfFf 344 lyophilized powder, and the production efficiency is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0050] Figure 1 This is a map of the E. coli inducible expression vector.

[0051] Figure 2 The supernatant and precipitate of pET30-BlfFf expression were detected by SDS-PAGE.

[0052] Figure 3 The figure shows the SDS-PAGE detection result of BlfF 344 dissolved in 8 M urea.

[0053] Figure 4 This is the gel filtration chromatogram of BlfFf 344.

[0054] Figure 5 The results of SDS-PAGE detection of bovine lactoferrin peptide BlfFf 344 after inclusion body solubilization and renaturation.

[0055] Figure 6 This is the antibacterial activity diagram of bovine lactoferrin peptide BlfFf 344 after renaturation. DETAILED DESCRIPTION

[0056] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0057] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0058] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0059] The technical principle of the present invention is as follows:

[0060] The present invention provides a method for preparing bovine lactoferrin peptide inclusion bodies, the preparation method comprising:

[0061] S1. Designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codon of Escherichia coli, and constructing an Escherichia coli inducible expression vector with the coding DNA;

[0062] S2. transforming the E. coli inducible expression vector into E. coli to obtain genetically engineered recombinant expression E. coli;

[0063] S3. Inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344 with isopropylthiogalactoside, the expression product forms bovine lactoferrin peptide inclusion bodies, and obtaining a bacterial mixture;

[0064] S4. The bacterial mixture is centrifuged to collect the Escherichia coli cells, which are resuspended and then broken, and then centrifuged to collect the bovine lactoferrin peptide inclusion bodies.

[0065] Step S1 specifically includes:

[0066] Designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codons of Escherichia coli, and homologously recombining the coding DNA with the linearized vector pET (containing T7 promoter + lactose operator) to obtain an Escherichia coli inducible expression vector;

[0067] Wherein, the amino acid sequence of the bovine lactoferrin peptide BlfFf 344 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding DNA is shown in SEQ ID NO.2;

[0068] The E. coli inducible expression vector map is as follows Figure 1 shown.

[0069] In the present invention, the advantage of designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codons of Escherichia coli is that the codons are optimized to correspond to the abundant aa-tRNA of the host bacteria, so that the protein polypeptide synthesis is carried out efficiently. The advantage of using the vector pET to construct the Escherichia coli inducible expression vector is that its expression system uses the viral promoter of T7 phage, which has high transcription efficiency. The vector uses the lactose operator as a regulatory element and can use IPTG (isopropylthiogalactoside) as a placebo inducer to effectively regulate the expression of the target gene.

[0070] The applicant found that the 344aa at the N-terminus of bovine lactoferrin contains the functional domains of Lfcin B and LFampin, and does not exhibit antibacterial activity against bacteria itself. However, after being decomposed by protease, the antibacterial functional domains Lfcin B and LFampin can be released. Therefore, the present invention selects the 344aa at the N-terminus of bovine lactoferrin shown in SEQ ID NO.1 as the target polypeptide, synthesizes its encoding gene according to the codon preference of the Escherichia coli expression system, constructs it into an inducible expression vector and transfers it into Escherichia coli cells to induce efficient expression of the target polypeptide.

[0071] Step S3 specifically includes:

[0072] The genetically engineered recombinant expression Escherichia coli is inoculated into a liquid culture medium. When the genetically engineered recombinant expression Escherichia coli grows to OD600=0.6±0.1, the culture temperature is controlled to be 25±1°C, isopropylthiogalactoside is added at a final concentration of 1±0.1 mmol / L to induce the efficient expression of the bovine lactoferrin peptide BlfFf 344, and the expression product forms bovine lactoferrin peptide inclusion bodies to obtain a bacterial mixed solution.

[0073] In the present invention, isopropyl thiogalactoside is used to induce the efficient expression of bovine lactoferrin peptide BlfFf 344. As a placebo-type inducer, isopropyl thiogalactoside is not degraded by cells and can continuously exert induction activity. The concentration of isopropyl thiogalactoside is 1±0.1mmol / L, which can ensure sufficient induction effect while reducing costs.

[0074] Step S4 specifically includes:

[0075] Centrifuging the bacterial cell mixture, removing the supernatant, and collecting the Escherichia coli cells;

[0076] The Escherichia coli cells are resuspended and then broken by ultrasonic wave to obtain inclusion body mixed solution;

[0077] The inclusion body mixture is centrifuged, the supernatant is removed, and the precipitate is collected to obtain bovine lactoferrin peptide inclusion bodies.

[0078] The present invention also provides a method for renaturing bovine lactoferrin peptide inclusion bodies, the method comprising:

[0079] 1) dissolving bovine lactoferrin peptide inclusion bodies in a urea solution to obtain a mixed solution;

[0080] 2) purifying the mixed solution by gel filtration chromatography to obtain a bovine lactoferrin peptide solution;

[0081] 3) adding a refolding solution to the bovine lactoferrin peptide solution and performing dialysis to obtain a bovine lactoferrin peptide refolding solution;

[0082] 4) freeze-drying the bovine lactoferrin peptide renaturation solution to obtain bovine lactoferrin peptide BlfFf 344.

[0083] Wherein, the bovine lactoferrin peptide inclusion body is prepared by the above-mentioned method for preparing bovine lactoferrin peptide inclusion body.

[0084] Step 1) specifically includes:

[0085] The bovine lactoferrin peptide inclusion bodies were washed and added to 8.0 mol / L urea solution, stirred and dissolved at 4±1°C for 2.0 h, and then ultrasonically assisted melting was performed until the solution became transparent to obtain a mixed solution;

[0086] Wherein, the ratio of the mass of the bovine lactoferrin peptide inclusion body to the volume of the urea solution is 1 g:10 mL.

[0087] In the present invention, the advantage of using urea solution to dissolve the bovine lactoferrin peptide inclusion bodies is that urea can effectively dissolve the inclusion bodies and has low cost. Too high or too low urea concentration can easily cause insufficient dissolution and result in loss of target protein.

[0088] In the present invention, the ratio of the mass of bovine lactoferrin peptide inclusion bodies to the volume of the urea solution is 1 g:10 mL, which can improve the dissolution effect and ensure the efficient preparation of the target protein.

[0089] Step 2) specifically includes:

[0090] The mixed solution was centrifuged, and the supernatant was collected and filtered through a 0.45 μm filter membrane, and then purified through a Super Tandex 75 prepgrade gel column to obtain a bovine lactoferrin peptide solution.

[0091] In the present invention, the purpose of passing the supernatant through a 0.45 μm filter membrane is to block cell debris and other larger cell impurities, to ensure that the inclusion bodies can be effectively filtered, and after being purified through a SuperTandex75prepgrade gel column, a single bovine lactoferrin peptide can be effectively purified.

[0092] Step 3) specifically includes:

[0093] Adding the refolding solution to the bovine lactoferrin peptide solution, placing the solution in a 14KDa dialysis bag for dialysis at a dialysis temperature of 4±1°C, replacing the dialysis solution once every 12 hours, and replacing the dialysis solution three times in total to obtain a bovine lactoferrin peptide refolding solution;

[0094] Wherein, the dialysate is 1XPBS buffer with a pH of 7.4 (NaCl: 137mM, KCl: 2.7mM, Na2HPO4: 10mM, KH2PO4: 2mM);

[0095] The volume ratio of the bovine lactoferrin peptide solution to the refolding solution is 1:100;

[0096] The composition and concentration of the solute in the refolding solution are as follows:

[0097] Tris.Cl: 50mmol / L, EDTA.Na2: 0.5mmol / L, NaCl: 50mmol / L, oxidized glutathione: 0.05mmol / L, glutathione: 0.5mmol / L, glycerol: 5%.

[0098] In the present invention, the volume ratio of the bovine lactoferrin peptide solution to the refolding solution is 1:100, which has the advantage of ensuring the most effective dissolution and refolding of the protein polypeptide in the inclusion body.

[0099] In the present invention, the advantage of using the above ingredients and concentrations in the refolding solution is that it has a more accurate pH buffer, the ratio of oxidized glutathione and glutathione is beneficial to the stability of the disulfide bonds of the polypeptide and the sulfhydryl groups of the cysteic acid residues, and a certain concentration of NaCl and glycerol is beneficial to the stability and activity maintenance of the polypeptide molecules in the solution.

[0100] The preparation method and renaturation method of bovine lactoferrin peptide inclusion bodies of the present application will be described in detail below in combination with examples and experimental data.

[0101] Example 1

[0102] This embodiment provides a method for preparing bovine lactoferrin peptide inclusion bodies.

[0103] 1. Construction of pET30-BlfFf expression vector

[0104] 1. Target sequence amplification

[0105] Using pUC-BlfFf vector (cloned with the designed and synthesized sequence shown in SEQ ID NO.2) as PCR template, Blf-f1 and Blf-r1 were selected as forward and reverse primers respectively for DNA polymerase chain reaction to obtain the BlfFf 344 gene fragment. The expected length of the target fragment was 1053 bp.

[0106] The specific sequences of primers Blf-f1 and Blf-r1 are shown in Table 1:

[0107] Table 1 Sequences of primers Blf-f1 and Blf-r1

[0108]

[0109] 2. Linearization of pET vector

[0110] The vector pET was double-digested with endonucleases XhoI and NdeI. The digestion system was: 1 μg pET plasmid DNA, 1 μL each of endonucleases XhoI and NdeI, 5 μL 10x CutSmart Buffer, and deionized water was used to make up to 50 μL, and the mixture was incubated at 37°C for 3 hours, and the enzymes were inactivated at 65°C for 30 minutes.

[0111] 3. Recombination reaction

[0112] The BlfFf 344 gene fragment and the linearized pET vector were recombined using the homologous recombination kit Vazyme The reaction product was transformed into E.coliDH5α, and the positive clones containing the target gene BlfFf 344 were screened using LB plates containing 50μg / mL kanamycin (Kan) and single colony PCR technology. Two positive clones were selected and cultured in LB culture medium for plasmid DNA extraction. After sequencing, the plasmids were preserved. The map of the preserved E. coli inducible expression vector is shown in the figure. Figure 1 As shown, it is referred to as pET30-BlfFf hereinafter.

[0113] 2. Transformation of pET30-BlfFf expression vector

[0114] 1. Prepare competent E. coli host bacteria BL21: Inoculate a single BL21 colony into a shake flask with rich medium for overnight culture, collect the bacteria by centrifugation at 1000 g, and then suspend them in 0.1 M CaCl2 solution and place them at 4°C overnight.

[0115] 2. Transformation of host bacteria: Take 0.1g of the prepared pET30-BlfFf plasmid and add it to the competent bacteria, vortex to mix, place it in an ice bath for 10 minutes, then place it in a 42°C water bath for 1 minute, and then quickly place it in an ice bath for 10 minutes.

[0116] 3. Plating and screening: Spread the bacteria onto a rich solid plate culture medium containing 100 μg / mL of ampicillin, invert and culture at 37°C overnight, and perform plasmid sequencing on the colonies to ensure their correctness.

[0117] 3. Inducible expression of pET30-BlfFf-BL21 recombinant bacteria

[0118] 1) Select a single positive bacterium and inoculate it into 50 mL LB liquid medium containing kan (50 μg / mL) and culture it at 37°C overnight.

[0119] 2) Then inoculate the overnight bacterial suspension into new LB medium at a ratio of 1:100 and culture until OD 600 = about 0.6, IPTG (1 mmol / L) was added, and the flask was shaken at 25°C and 180 r / min for 6 h to induce the expression of the target protein polypeptide.

[0120] 3) After induction, centrifuge at 6000 r / min for 10 min at room temperature, remove the supernatant and collect the bacteria.

[0121] 4) Take 2 mL of culture medium into a centrifuge tube and perform SDS-PAGE for small amount of protein expression to understand the expression of inclusion bodies.

[0122] 5) Add 2 mL of collected bacteria to 1×PBS (NaCl: 137mM, KCl: 2.7mM, Na2HPO4: 10mM, KH2PO4: 2mM, pH=7.2) to resuspend the precipitate and perform ultrasonic disruption. After disruption, centrifuge the sample at a speed of 12000r / min to separate the supernatant and precipitate. Add 5×loading buffer to the supernatant and precipitate in a ratio of 4:1 and mix thoroughly. Boil the sample for 10 minutes and perform SDS-PAGE gel detection.

[0123] 6) The sample volume for each well was 10 μL. The concentration gel was run at 80 V for 20 min, and the separation gel was run at 120 V for 80 min.

[0124] 7) Coomassie Brilliant Blue staining, observation and photography (such as Figure 2 ).

[0125] Figure 2 In the figure, M: molecular weight standard; 1: uninduced pET30-BlfFf supernatant; 2: IPTG-induced pET30 supernatant (empty vector control); 3: IPTG-induced pET30-BlfFf supernatant; 4: uninduced pET30-BlfFf precipitate; 5: IPTG-induced pET precipitate (empty vector control); 6: IPTG-induced pET30-BlfFf precipitate. The results showed that the induced expressed BlfFf 344 only appeared in the precipitate of broken bacteria, indicating that they were all insoluble inclusion bodies. In order to obtain functional polypeptides, they need to be purified, dissolved and renatured.

[0126] Example 2

[0127] This embodiment provides a method for renaturing bovine lactoferrin peptide inclusion bodies.

[0128] 1. Dissolution of BlfFf 344 inclusion bodies

[0129] 1. Use 10 pre-weighed 50 mL centrifuge tubes to dispense 500 mL of the fermentation broth obtained in step 2) of the third part of Example 1, centrifuge at 6000 r / min, 4°C for 10 min, discard the supernatant, and collect the bacteria.

[0130] 2. Weigh the bacteria, and resuspend them in the ratio of 0.1 g of bacteria (wet weight) to 1 mL of 1xPBS (NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, KH2PO4: 2 mM, pH = 7.2) buffer solution (4°C). Use a pipette to slowly pipette and mix, then centrifuge at 6000 r / min, 4°C for 10 min, and repeat the suspension and centrifugation to wash the bacteria twice.

[0131] 3.Use ultrasonic to disrupt the washed cells, add 1 mL 1xPBS buffer solution (4°C) to 0.1 g of cells and resuspend the cells, and use a pipette to mix them. The disruption conditions are: 320 W ultrasonic power, 2 seconds of ultrasonic disruption, 3 seconds of interval, and a total of 20 minutes. After the disruption is completed, centrifuge at 12000 r / min, 4°C for 15 minutes to collect the cell precipitation.

[0132] 4. Add 1 mL of 2% Triton X-100 (polyethylene glycol octylphenyl ether) washing buffer for every 0.1 g of bacterial cells to wash the precipitate, centrifuge at 12000 r / min for 15 min to collect the inclusion bodies, repeat the washing and centrifugation twice to remove the supernatant.

[0133] 5. Add the cleaned inclusion bodies to the inclusion body dissolution solution (8M urea) for dissolution. Add 1mL of the inclusion body dissolution solution to 0.1g of the cells and stir and dissolve in a magnetic stirrer at 4°C for 2h. Then, ultrasonically dissolve the dissolved inclusion body solution until the solution becomes transparent. The ultrasonic treatment conditions are: 130W ultrasonic power, ultrasonic fragmentation for 5s, interval of 2s, and a total time of 2min.

[0134] 6. Centrifuge the dissolved inclusion body solution at 12000r / min and 4℃ for 10min, collect the supernatant and place it in an ice water bath to obtain the protein solution.

[0135] 7. Perform SDS-PAGE electrophoresis analysis, the results Figure 3 As shown, where M: molecular weight standard; 1: BlfFf 344 dissolved in 8 M urea.

[0136] 2. Purification of peptide BlfFf 344

[0137] 1. This example uses gel filtration chromatography to purify the protein. According to the molecular weight of the protein, we selected SuperTandex 75prep grade gel column.

[0138] 2. Column loading: ① Check the chromatography column to ensure that all parts are complete and clean. Install the lower column head, tighten the O-ring, and fix the chromatography column vertically on the iron stand to keep the chromatography column horizontal. ② Add an appropriate amount of column loading solution to the chromatography column, leaving a space of 1-2 cm in height in the column, and pour the column solution into the chromatography column continuously to avoid bubbles. ③ Connect the upper column head to the low-pressure chromatography system, turn on the pump at a certain flow rate to discharge the residual air in the pipeline. At this time, the medium and solution are separated, and the supernatant solution is completely clarified. ④ Tighten the O-ring and turn on the pump at a certain flow rate until the column bed volume no longer changes, and mark the interface. ⑤ Turn off the pump, there are no bubbles, the column volume no longer changes, and the column loading is complete.

[0139] 3. Experimental process: ① Balance: Use deionized water to balance the column for 5-10 column volumes before loading, until the recorder baseline becomes stable and the value is 0. ② Loading: Filter the protein solution with a 0.45μm filter membrane before loading. ③ Elution: Elute with deionized water, keep the flow rate (1mL / min) and buffer composition unchanged during elution. ④ Collect protein solution according to the peak value ( Figure 4 ).

[0140] Figure 4 The gel filtration chromatography spectrum of bovine lactoferrin peptide BlfFf 344 is shown, and the elution peak (BlfFf) is obtained. The gel filtration chromatography filler is SuperTandex G75.

[0141] 3. Renaturation of peptide BlfFf 344

[0142] 1. The protein solution sample purified by gel filtration chromatography was placed in a dialysis bag for dialysis. In this embodiment, a 14KDa dialysis bag was used.

[0143] 2. Treatment of dialysis bag: ① According to the dialysis sample volume and the unit length of the dialysis bag, cut the dialysis bag of appropriate length. ② Immerse the dialysis bag in a container containing 500mL of dialysis bag treatment solution (2% NaHCO3, 1mM EDTA, pH8.0), and place the dialysis bag in a boiling water bath for 10 minutes. ③ Thoroughly wash the dialysis bag after the boiling water bath treatment with distilled water. ④ Place the dialysis bag in a 1mM EDTA solution and treat it in a boiling water bath for another 10 minutes. ⑤ After cooling, transfer to distilled water or 20% ethanol and place at 4°C for use.

[0144] 3.Put the sample solution into a dialysis bag, add the dialysis bag into the refolding solution (50mM Tris.Cl, 0.5mM EDTA.Na2, 50mM NaCl, 0.05mM oxidized glutathione GSSG, 0.5mM glutathione GSH, 5% glycerol) at a ratio of 1:100, and replace with fresh 1xPBS dialysis solution every 12 hours at 4°C, for a total of three changes.

[0145] 4. Transfer the dialyzed BlfFf 344 protein solution to a 50 mL centrifuge tube and freeze-dry it for 12-24 hours into powder.

[0146] IV. SDS-PAGE detection of dissolved and refolded BlfFf 344

[0147] 1. Add 1 / 5 volume of 5× loading buffer, mix thoroughly, boil the sample for 10 min, and perform SDS-PAGE gel detection.

[0148] 2. The sample volume for each well is 10 μL. Run the concentration gel at 80V for 20 minutes and the separation gel at 120V for 80 minutes.

[0149] 3. Coomassie Brilliant Blue staining, observation and photography ( Figure 5 ).

[0150] Figure 5 This is the SDS-PAGE of BlfF 344 after inclusion body solubilization, where M is a protein molecular weight standard; 1 is a dissolved and renatured BlfFf 344 sample (dialysis product obtained in step 3, 3); 2 is a freeze-dried BlfFf 344 sample (10-fold dilution).

[0151] According to the test, in the above process of this embodiment, the wet weight of the collected bacteria in 1L of fermentation broth is 5-8g, and the dry weight is 0.5-0.8g. 100-150mg of refolded bovine lactoferrin freeze-dried powder can be purified from 1L of fermentation broth.

[0152] Example 3

[0153] In this example, the antibacterial performance of the renatured polypeptide BlfFf 344 prepared in Example 2 was tested.

[0154] 1. Activate E. coli DH5α by streaking, pick a single colony and culture it in LB medium. Resuspend the bacterial solution in physiological saline and dilute it to 10% 8 CFU / mL, the corresponding bacterial solution OD 600 =about 0.6.

[0155] 2. Heat 50 mL of solid culture medium until the temperature is appropriate, add a certain amount of DH5α bacterial solution, shake well, and pour into the culture dish.

[0156] 3. After the culture medium solidified, holes were punched in the culture medium, and protease-treated BlfFf344 protein samples and antibiotics were added as positive controls, and BlfFf344 protein samples not treated with protease were added as negative controls.

[0157] 4. Protein treatment: Add 1% pepsin to the protein solution at a volume ratio of 1 / 30 and perform enzymatic hydrolysis at 37°C for 30 minutes.

[0158] 5. Place the plate with the sample in an incubator, incubate overnight at 37°C, and observe and take photos ( Figure 6 ).

[0159] Figure 6In the figure, a: BlfFf 344 treated with pepsin showed antibacterial activity against Escherichia coli; b: 50 μg / mL Amp (ampicillin) solution control; c: BlfFf 344 protein without protease treatment had no antibacterial activity; d: 10 mg / mL pepsin solution control. Figure 6 It can be seen that the polypeptide BlfFf 344 prepared by dissolution and renaturation of inclusion bodies showed complete polypeptide activity, could be decomposed by pepsin and release antibacterial peptide segments, and its antibacterial activity was equivalent to that of 50 μg / mL ampicillin.

[0160] In summary, the present invention induces the expression of the constructed target bovine lactoferrin peptide BlfFf 344 through E. coli shake flask fermentation, and the BlfFf 344 products are all insoluble inclusion bodies; the present invention takes advantage of the convenience of preparation of inclusion bodies, and can collect inclusion bodies and remove a large amount of impurities through centrifugation. The present invention establishes an effective dissolution process for inclusion bodies, and after dissolution, the inclusion bodies are subjected to SuperTandex 75prep grade molecular sieve chromatography, which can effectively purify them, and establishes renaturation conditions for the dissolved and purified polypeptides, and can efficiently prepare BlfFf 344 with functional activity.

[0161] In the present invention, the amino acid sequence of bovine lactoferrin peptide BlfFf 344 is as follows:

[0162] MKLFVPALLSLGALGLCLAAPRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIPGIAEKKADAVTLDGGMVFEAGRDPYKLRPVAAEIYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCHTGLGRSAGWIIPMGILRPYLSWTESLEPLQGAVAKFF SASCVPCIDRQAYPNLCQLCKGEGENQCACSSREPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNSRAPVDAFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQEKSGKNKSRSFQLFGSPPGQRDLLFKDSALGFLRIPSKVDSALYLGSRYL.

[0163] The codon-optimized BlfFf 344 gene sequence in the E. coli expression host is as follows (including 6XHis):

[0164]

[0165] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0166] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0167] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing bovine lactoferrin peptide inclusion bodies, characterized in that: The preparation method comprises: Designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codons of Escherichia coli, and constructing an Escherichia coli inducible expression vector with the coding DNA; Transforming the E. coli inducible expression vector into E. coli to obtain genetically engineered recombinant expression E. coli; Inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344 with isopropylthiogalactoside, and the expression product forms bovine lactoferrin peptide inclusion bodies to obtain a bacterial cell mixture; The bacterial mixture is centrifuged to collect the Escherichia coli cells, which are resuspended and then broken, and then centrifuged to collect the bovine lactoferrin peptide inclusion bodies.

2. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, characterized in that: The coding DNA of bovine lactoferrin peptide BlfFf 344 is designed based on the preferred codons of Escherichia coli, and the Escherichia coli inducible expression vector is constructed with the coding DNA, which specifically includes: Designing the coding DNA of bovine lactoferrin peptide BlfFf 344 based on the preferred codons of Escherichia coli, and homologously recombining the coding DNA with the linearized vector pET to obtain an Escherichia coli inducible expression vector; Wherein, the amino acid sequence of the bovine lactoferrin peptide BlfFf 344 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding DNA is shown in SEQ ID NO.2; The map of the E. coli inducible expression vector is shown in FIG1 .

3. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, characterized in that: The step of transforming the E. coli inducible expression vector into E. coli to obtain genetically engineered recombinant expression E. coli specifically includes: The E. coli inducible expression vector is transformed into E. coli BL21 to obtain genetically engineered recombinant expression E. coli.

4. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, characterized in that: The method of inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf344 with isopropylthiogalactoside, wherein the expression product forms bovine lactoferrin peptide inclusion bodies, and obtaining a bacterial cell mixture specifically comprises: The genetically engineered recombinant expression Escherichia coli is inoculated into a liquid culture medium. When the genetically engineered recombinant expression Escherichia coli grows to OD600=0.6±0.1, the culture temperature is controlled to be 25±1°C, isopropylthiogalactoside is added at a final concentration of 1±0.1 mmol / L to induce the efficient expression of the bovine lactoferrin peptide BlfFf 344, and the expression product forms bovine lactoferrin peptide inclusion bodies to obtain a bacterial mixed solution.

5. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, characterized in that: The method comprises centrifuging the bacterial mixture to collect the Escherichia coli cells, resuspending the cells and breaking the bacterial wall, and then centrifuging to collect the bovine lactoferrin peptide inclusion bodies, which specifically includes: Centrifuging the bacterial cell mixture, removing the supernatant, and collecting the Escherichia coli cells; The Escherichia coli cells are resuspended and then broken by ultrasonic treatment to obtain an inclusion body mixed solution; The inclusion body mixture is centrifuged, the supernatant is removed, and the precipitate is collected to obtain bovine lactoferrin peptide inclusion bodies.

6. A method for renaturing bovine lactoferrin peptide inclusion bodies, characterized in that: The method comprises: dissolving bovine lactoferrin peptide inclusion bodies in a urea solution to obtain a mixed solution; The mixed solution is purified by gel filtration chromatography to obtain a bovine lactoferrin peptide solution; adding a refolding solution to the bovine lactoferrin peptide solution and performing dialysis to obtain a bovine lactoferrin peptide refolding solution; The bovine lactoferrin peptide renaturation solution was freeze-dried to obtain bovine lactoferrin peptide BlfFf 344. Wherein, the bovine lactoferrin peptide inclusion body is prepared by the method for preparing bovine lactoferrin peptide inclusion body according to any one of claims 1 to 5.

7. A method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 6, characterized in that: The step of dissolving the bovine lactoferrin peptide inclusion bodies in a urea solution to obtain a mixed solution specifically comprises: The bovine lactoferrin peptide inclusion bodies were washed and added to 8.0 mol / L urea solution, stirred and dissolved at 4±1°C for 2.0 h, and then ultrasonically assisted melting was performed until the solution became transparent to obtain a mixed solution; Wherein, the ratio of the mass of the bovine lactoferrin peptide inclusion body to the volume of the urea solution is 1 g:10 mL.

8. A method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 6, characterized in that: The mixed solution is purified by gel filtration chromatography to obtain a bovine lactoferrin peptide solution, which specifically comprises: The mixed solution was centrifuged, the supernatant was collected and filtered through a 0.45 μm filter membrane, and then purified through a SuperTandex 75 prepgrade gel column to obtain a bovine lactoferrin peptide solution.

9. A method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 6, characterized in that: The step of adding a refolding solution to the bovine lactoferrin peptide solution and then dialyzing the solution to obtain the bovine lactoferrin peptide refolding solution specifically comprises: Adding the refolding liquid to the bovine lactoferrin peptide solution, and then filling it into a 14KDa dialysis bag for dialysis to obtain a bovine lactoferrin peptide refolding solution; The volume ratio of the bovine lactoferrin peptide solution to the refolding solution is 1:100; The composition and concentration of the solute in the refolding solution are as follows: Tris.Cl: 50mmol / L, EDTA.Na2: 0.5mmol / L, NaCl: 50mmol / L, oxidized glutathione: 0.05mmol / L, glutathione: 0.5mmol / L, glycerol: 5%.

10. A method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 9, characterized in that: The step of adding a refolding liquid to the bovine lactoferrin peptide solution and then placing the solution in a 14KDa dialysis bag for dialysis to obtain a bovine lactoferrin peptide refolding solution specifically comprises: Adding the refolding solution to the bovine lactoferrin peptide solution, placing the solution in a 14KDa dialysis bag for dialysis at a dialysis temperature of 4±1°C, replacing the dialysis solution once every 12 hours, and replacing the dialysis solution three times in total to obtain a bovine lactoferrin peptide refolding solution; Wherein, the dialysate is 1XPBS buffer with a pH of 7.4.

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