Preparation method and renaturation method of bovine lactoferrin peptide inclusion bodies

Abstract

The present invention provides a preparation method and renaturation method of bovine lactoferrin peptide inclusion bodies, which belong to the field of biotechnology. The preparation method includes: designing the coding DNA of bovine lactoferrin peptide Bl fFf 344 based on the preferred codon of Escherichia coli, then constructing an Escherichia coli inducible expression vector and transforming it into Escherichia coli to obtain genetically engineered recombinant expression Escherichia coli; inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide Bl fFf344 and form inclusion bodies with isopropylthiogalactoside to obtain a bacterial mixture; centrifuging the bacterial mixture to collect the Escherichia coli cells, resuspending them and performing a cell wall breaking process, and then centrifuging to collect the bovine lactoferrin peptide inclusion bodies. The method can efficiently prepare bovine lactoferrin peptide inclusion bodies, and combined with the bovine lactoferrin peptide inclusion body separation, dissolution, purification and renaturation method established by the present invention, realizes genetic engineering and efficient production of bovine lactoferrin peptide.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for preparing and refolding bovine lactoferrin peptide inclusion bodies. Background Art

[0002] Expressing exogenous genes using *E. coli* is a major method for producing proteins and peptides through genetic engineering. *E. coli* is easy to ferment and culture and has a well-developed induction and regulation expression system, making it the primary system for producing exogenous proteins through genetic engineering. Compared to other expression systems such as yeast, plant cell, and insect cell systems, it exhibits unique advantages in simplicity and efficiency, and is currently widely used in the genetic engineering production of drugs such as human insulin, human growth hormone, and erythropoietin (EPO).

[0003] The biggest challenge in expressing exogenous proteins using *E. coli* is that when these proteins are synthesized in large quantities within *E. coli* cells, they readily aggregate into insoluble protein aggregates—inclusion bodies. Inclusion body formation often prevents the target peptide from effectively dissolving and thus fails to exhibit activity, leading to production failure. However, if effective dissolution and refolding conditions are established for inclusion bodies of a specific protein peptide in genetic engineering expression, allowing them to dissolve and restore their correct conformation and normal function, then inclusion body expression can actually facilitate protein peptide purification and improve production efficiency.

[0004] Bovine lactoferrin (LF) is an 80 kDa iron-binding glycoprotein found in bovine exocrine secretions (milk) and certain granules of polymorphonuclear lymphocytes. Besides its antibacterial activity, it regulates various biological functions, including iron transfer, anti-tumor activity, and viral activity. It also participates in anti-infection and immune processes by regulating various cytokines. Bovine lactoferrin is a novel antimicrobial peptide and a promising alternative to antibiotics in animal husbandry. Drago-Serrano et al. fed different doses of bovine lactoferrin to rats infected with Salmonella typhimurium and found that bovine lactoferrin reduced the number of Salmonella typhimurium in the rats. The antibacterial activity of bovine lactoferrin is actually mediated by the foliated structure of the bovine lactoferrin functional fragment (BlfFf). Bovine lactoferrin produces two small peptides after protease hydrolysis, namely the 17-41 aa bovine lactoferrin peptide (Lfcin B) and the 268-284 aa bovine lactoferrin peptide (LF ampin) domains located at the N-terminus.

[0005] Numerous reports and patents exist regarding the genetic engineering expression of bovine lactoferrin peptides. 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*, and the expressed protein existed in the form of inclusion bodies. The purified Lfcin B recombinant protein inclusion bodies were renatured using TGE dialysis buffer. 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, obtaining inactive or non-toxic bovine lactoferrin peptide precursors. Yeast expression systems have been applied to the expression of bovine lactoferrin. Cha Xiangdong et al. (CN104073511A) constructed and prepared constitutive expression vectors for yeast. Li Bentao et al. (CN103602603A) obtained a patent for expressing and preparing bovine lactoferrin peptides in Pichia pastoris. Zhang Guanghua et al. (CN117004501B) secreted and expressed bovine lactoferrin antimicrobial peptides in Pichia pastoris. Wang Jianhua et al. (CN102408480A) also disclosed a patent entitled "A yeast-expressed bovine lactoferrin-derived antimicrobial fragment BLfA and its preparation method." However, existing technologies for expressing bovine lactoferrin peptides often result in the target protein peptide inhibiting the growth of the host bacteria, and the purification efficiency of the protein product is relatively low, leading to relatively high manufacturing costs. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing bovine lactoferrin peptide inclusion bodies. This method can efficiently prepare bovine lactoferrin peptide inclusion bodies, which have almost no impact on the growth of host bacteria and are easy to purify. This significantly improves the production efficiency of bovine lactoferrin peptides. Combined with the established methods for the separation, dissolution, purification, and refolding of bovine lactoferrin peptide inclusion bodies, this invention enables the efficient production of bovine lactoferrin peptides through genetic engineering.

[0007] The present 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] The E. coli inducible expression vector was transformed into E. coli to obtain genetically engineered recombinant E. coli;

[0012] Isopropyl thiogalactoside was used to induce the genetically engineered recombinant Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344. The expression product formed bovine lactoferrin peptide inclusion bodies, and a bacterial cell mixture was obtained.

[0013] The bacterial mixture was centrifuged to collect Escherichia coli cells, which were then resuspended and subjected to cell disruption treatment. The bovine lactoferrin peptide inclusion bodies were then collected by centrifugation.

[0014] Furthermore, the design of the encoding DNA for bovine lactoferrin peptide BlfFf 344 based on E. coli preferred codons, and the construction of an E. coli inducible expression vector using the encoding DNA, specifically includes:

[0015] The encoding DNA of bovine lactoferrin peptide BlfFf 344 was designed based on the preferred codons of E. coli. The encoding DNA was homologously recombined with the linearized vector pET to obtain the E. coli inducible expression vector.

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

[0017] The map of the E. coli inducible expression vector 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 *E. coli* specifically includes:

[0019] The Escherichia coli inducible expression vector was transformed into Escherichia coli BL21 to obtain genetically engineered recombinant expressed Escherichia coli.

[0020] Furthermore, the method of inducing the recombinant Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344 using isopropyl thiogalactoside, with the expression product forming bovine lactoferrin peptide inclusion bodies, to obtain a bacterial cell mixture specifically includes:

[0021] The genetically engineered recombinant Escherichia coli was inoculated into a liquid culture medium. When the genetically engineered recombinant Escherichia coli grew to OD600 = 0.6 ± 0.1, the culture temperature was controlled at 25 ± 1℃, and isopropyl thiogalactoside was added at a final concentration of 1 ± 0.1 mmol / L to induce the efficient expression of the bovine lactoferrin peptide BlfFf 344. The expression product formed bovine lactoferrin peptide inclusion bodies, and a bacterial cell mixture was obtained.

[0022] Furthermore, the centrifugation of the bacterial mixture to collect E. coli cells, resuspending them and then performing cell wall disruption treatment, followed by centrifugation to collect the bovine lactoferrin peptide inclusion bodies, specifically includes:

[0023] The bacterial mixture was centrifuged, the supernatant was removed, and the Escherichia coli cells were collected.

[0024] The Escherichia coli cells were resuspended and then subjected to ultrasonic cell disruption to obtain an inclusion body mixture.

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

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

[0027] Bovine lactoferrin peptide inclusion bodies were dissolved in urea solution to obtain a mixed solution;

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

[0029] After adding a refolding solution to the bovine lactoferrin peptide solution, the solution was dialyzed to obtain a bovine lactoferrin peptide refolding solution.

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

[0031] The bovine lactoferrin peptide inclusion bodies are prepared by the above-mentioned method for preparing bovine lactoferrin peptide inclusion bodies.

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

[0033] After washing the bovine lactoferrin peptide inclusion bodies, they were added to an 8.0 mol / L urea solution and stirred at 4±1℃ for 2.0 h to dissolve. Then, ultrasonic dissolution was performed until the solution became transparent, thus obtaining a mixed solution.

[0034] The mass ratio of the bovine lactoferrin peptide inclusion bodies 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, specifically comprising:

[0036] The mixture was centrifuged, the supernatant was collected and filtered through a 0.45 μm filter membrane, and then purified by SuperTandex 75 prep grade gel column to obtain bovine lactoferrin peptide solution.

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

[0038] After adding refolding solution to the bovine lactoferrin peptide solution, the solution was filled into a 14 kDa dialysis bag and dialyzed 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: 50 mmol / L, EDTA.Na2: 0.5 mmol / L, NaCl: 50 mmol / L, Oxyglutathione: 0.05 mmol / L, Glutathione: 0.5 mmol / L, Glycerol: 5% (by weight).

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

[0043] After adding refolding solution to the bovine lactoferrin peptide solution, the solution was placed in a 14 kDa dialysis bag for dialysis at a temperature of 4 ± 1 °C. The dialysis solution was changed every 12 hours for a total of 3 times to obtain a bovine lactoferrin peptide refolding solution.

[0044] The dialysate is a 1X PBS 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. This invention discloses a method for preparing bovine lactoferrin peptide inclusion bodies. The method selects the N-terminal 344aa of bovine lactoferrin as the target peptide. First, based on the codon preference of the *E. coli* expression system, the encoding DNA of bovine lactoferrin peptide BlfFf 344 is designed. An *E. coli* inducible expression vector is constructed using this encoding DNA. This expression vector is transformed into *E. coli*, and IPTG (isopropyl galactothioglycoside) is used to induce efficient expression of the recombinant gene in engineered *E. coli*. The expression product forms insoluble bovine lactoferrin peptide inclusion bodies. *E. coli* cells are collected and lysed to obtain the bovine lactoferrin peptide inclusion bodies. This method, through optimized design of the encoding DNA codons and the use of the *E. coli* expression system for inclusion body expression, enables efficient expression of bovine lactoferrin peptide without affecting the growth of the host bacteria. The inclusion bodies formed by the expression product facilitate the purification of bovine lactoferrin peptide BlfFf 344, improving the efficiency of genetically engineered production of functional bovine lactoferrin peptides.

[0047] 2. This invention discloses a method for preparing bovine lactoferrin peptide inclusion bodies. The method expresses bovine lactoferrin functional peptides using inclusion body expression in *E. coli*. The bovine lactoferrin peptide BlfFf 344 contains two functional domains: LF ampin and Lfcin B. Experiments show that the inclusion bodies prepared in this invention, after dissolution and refolding, yield bovine lactoferrin peptide BlfFf344 exhibiting complete peptide activity. Upon treatment with pepsin, it can release the antimicrobial peptides LF ampin and Lfcin B.

[0048] 3. This invention provides a method for refolding bovine lactoferrin peptide inclusion bodies. The method involves dissolving bovine lactoferrin peptide inclusion bodies using a certain concentration of urea under established physical conditions. The resulting mixed solution is purified by gel filtration chromatography to obtain a single bovine lactoferrin peptide. A refolding solution is added during the dialysis purification process. The purified bovine lactoferrin peptide BlfFf 344 exhibits complete polypeptide activity. After treatment with pepsin, it releases the antimicrobial peptides LF ampin and Lfcin B. Experiments have shown that, combined with the preparation and refolding methods of bovine lactoferrin peptide inclusion bodies of this invention, 1L of culture medium containing recombinant Escherichia coli (wet weight 5-8g, dry weight 0.5-0.8g) can produce 100-150mg of lyophilized active bovine lactoferrin peptide BlfFf 344 powder, demonstrating high production efficiency. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0051] Figure 2 The supernatant and precipitate were used for SDS-PAGE detection of pET30-BlfFf expression.

[0052] Figure 3 The results of SDS-PAGE analysis of BlfF 344 dissolved in 8M urea.

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

[0054] Figure 5 The results of SDS-PAGE analysis of bovine lactoferrin peptide BlfFf 344 after inclusion body dissolution and refolding are shown.

[0055] Figure 6 This is a graph showing the antibacterial activity of the refolded bovine lactoferrin peptide BlfFf 344. Detailed Implementation

[0056] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0057] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

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

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

[0060] This invention discloses a method for preparing bovine lactoferrin peptide inclusion bodies, the method comprising:

[0061] S1. Design the encoding DNA of bovine lactoferrin peptide BlfFf 344 based on Escherichia coli preferred codons, and construct an Escherichia coli inducible expression vector using the encoding DNA;

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

[0063] S3. The genetically engineered recombinant Escherichia coli was induced to express the bovine lactoferrin peptide BlfFf 344 efficiently with isopropyl thiogalactoside. The expression product formed bovine lactoferrin peptide inclusion bodies, and a bacterial cell mixture was obtained.

[0064] S4. Centrifuge the bacterial mixture to collect Escherichia coli cells, resuspend them, and then perform cell disruption treatment. Finally, centrifuge to collect the bovine lactoferrin peptide inclusion bodies.

[0065] Step S1 specifically includes:

[0066] The encoding DNA of bovine lactoferrin peptide BlfFf 344 was designed based on the E. coli preferred codon. The encoding DNA was then homologously recombined with the linearized vector pET (which contains the T7 promoter and lactose operator) to obtain the E. coli inducible expression vector.

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

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

[0069] In this invention, the advantage of designing the encoding DNA of bovine lactoferrin peptide BlfFf 344 based on E. coli preferred codons is that it optimizes the abundant aa-tRNA of the host bacteria corresponding to the codons, enabling efficient protein and polypeptide synthesis. The advantage of using the pET vector to construct the E. coli inducible expression vector is that its expression system uses the T7 phage viral promoter, which has high transcription efficiency. The vector uses the lactose operator as a regulatory element and can effectively regulate the expression of the target gene using IPTG (isopropyl thiogalactoside) as a placebo inducer.

[0070] The applicant discovered that the N-terminal 344aa of bovine lactoferrin contains the functional domains Lfcin B and LFampin. While it does not exhibit antibacterial activity against bacteria, it can release the antibacterial functional domains Lfcin B and LFampin after proteolysis. Therefore, this invention selects the N-terminal 344aa of bovine lactoferrin shown in SEQ ID NO.1 as the target polypeptide, synthesizes its encoding gene according to the codon preference of the E. coli expression system, constructs it into an inducible expression vector, and transforms it into E. coli cells to induce efficient expression of the target polypeptide.

[0071] Step S3 specifically includes:

[0072] The genetically engineered recombinant Escherichia coli was inoculated into a liquid culture medium. When the genetically engineered recombinant Escherichia coli grew to OD600 = 0.6 ± 0.1, the culture temperature was controlled at 25 ± 1℃, and isopropyl thiogalactoside was added at a final concentration of 1 ± 0.1 mmol / L to induce the efficient expression of the bovine lactoferrin peptide BlfFf 344. The expression product formed bovine lactoferrin peptide inclusion bodies, and a bacterial cell mixture was obtained.

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

[0074] Step S4 specifically includes:

[0075] The bacterial mixture was centrifuged, the supernatant was removed, and the Escherichia coli cells were collected.

[0076] The Escherichia coli cells were resuspended and then subjected to ultrasonic cell disruption to obtain an inclusion body mixture.

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

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

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

[0080] 2) The mixed solution was purified by gel filtration chromatography to obtain bovine lactoferrin peptide solution;

[0081] 3) Add refolding solution to the bovine lactoferrin peptide solution and then dialyze to obtain a bovine lactoferrin peptide refolding solution;

[0082] 4) The bovine lactoferrin peptide refolding solution was freeze-dried to obtain bovine lactoferrin peptide BlfFf 344.

[0083] The bovine lactoferrin peptide inclusion bodies are prepared by the above-mentioned method for preparing bovine lactoferrin peptide inclusion bodies.

[0084] Step 1) specifically includes:

[0085] After washing the bovine lactoferrin peptide inclusion bodies, they were added to an 8.0 mol / L urea solution and stirred at 4±1℃ for 2.0 h to dissolve. Then, ultrasonic dissolution was performed until the solution became transparent, thus obtaining a mixed solution.

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

[0087] In this invention, the advantage of using urea solution to dissolve bovine lactoferrin peptide inclusion bodies is that urea can effectively dissolve inclusion bodies and is relatively inexpensive. Too high or too low urea concentration can easily lead to insufficient dissolution and loss of the target protein.

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

[0089] Step 2) specifically includes:

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

[0091] In this invention, the purpose of passing the supernatant through a 0.45μm filter membrane is to block cell debris and other larger cellular impurities, ensuring that inclusion bodies can be effectively filtered out. After purification by SuperTandex 75 prepgrade gel column, a single bovine lactoferrin peptide can be effectively obtained.

[0092] Step 3) specifically includes:

[0093] After adding refolding solution to the bovine lactoferrin peptide solution, the solution was placed in a 14 kDa dialysis bag for dialysis at a temperature of 4 ± 1 °C. The dialysis solution was changed every 12 hours for a total of 3 times to obtain a bovine lactoferrin peptide refolding solution.

[0094] 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: 50 mmol / L, EDTA.Na2: 0.5 mmol / L, NaCl: 50 mmol / L, Oxyglutathione: 0.05 mmol / L, Glutathione: 0.5 mmol / L, Glycerol: 5%.

[0098] In this invention, the advantage of using a volume ratio of bovine lactoferrin peptide solution to refolding solution of 1:100 is that it ensures the most effective dissolution and refolding of the protein peptides in the inclusion bodies.

[0099] In this invention, the advantage of using the above-mentioned components and concentrations in the refolding solution is that it has a more precise pH buffer, the ratio of oxidized glutathione and glutathione is conducive to the stability of the disulfide bonds and sulfhydryl groups of cysteine ​​residues in the polypeptide, and a certain concentration of NaCl and glycerol is conducive to the stability and activity maintenance of polypeptide molecules in solution.

[0100] The preparation and refolding methods of bovine lactoferrin peptide inclusion bodies of this application will be described in detail below with reference to embodiments and experimental data.

[0101] Example 1

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

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

[0104] 1. Amplification of the target sequence

[0105] Using the pUC-BlfFf vector (cloned with the designed and synthesized sequence shown in SEQ ID NO.2) as a PCR template, Blf-f1 and Blf-r1 were used as forward and reverse primers, respectively, to perform 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. Primer sequences Blf-f1 and Blf-r1

[0108]

[0109] 2. Linearized carrier pET

[0110] The pET vector was double-digested using the restriction enzymes XhoI and NdeI. The digestion system consisted of 1 μg pET plasmid DNA, 1 μL each of XhoI and NdeI restriction enzymes, 5 μL of 10x CutSmart Buffer, and deionized water to a final volume of 50 μL. The mixture was incubated at 37°C for 3 h and then the enzymes were inactivated at 65°C for 30 min.

[0111] 3. Recombination reaction

[0112] The BlfFf 344 gene fragment and the linearized pET vector were combined using the Vazyme homologous recombination kit. The reaction product was transformed into E. coli DH5α using the Ultra One Step Cloning Kit and incubated at 50°C for 30 minutes. Positive clones containing the target gene BlfFf 344 were screened using LB plates containing 50 μg / mL kanamycin (Kan) and single colony PCR. Two positive clones were selected and expanded 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 will be referred to as pET30-BlfFf below.

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

[0114] 1. Preparation of competent Escherichia coli host strain BL21: After inoculating a single colony of BL21 into a shake flask containing rich medium and culturing overnight, the bacteria were collected by centrifugation at 1000g and then suspended in 0.1M CaCl2 solution and incubated overnight at 4°C.

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

[0116] 3. Plating and screening: The bacteria were spread onto rich solid agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C overnight. The colonies that were plasmidized were identified by plasmid sequencing to ensure their accuracy.

[0117] III. Induced expression of pET30-BlfFf-BL21 recombinant bacteria

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

[0119] 2) The overnight bacterial culture was then inoculated into fresh LB medium at a ratio of 1:100 and cultured until OD500 was reached. 600 = Approximately 0.6, add IPTG (1 mmol / L) and culture in a shake flask at 25℃ and 180 r / min for 6 h to induce expression of the target protein peptide.

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

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

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

[0123] 6) Load 10 μL of sample into each well, run the stacking gel at 80V for 20 min, and run the separating gel at 120V for 80 min.

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

[0125] Figure 2 In the table, M represents the 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 BlfFf 344 only appeared in the precipitate of the lysed bacteria, indicating that it was an insoluble inclusion body. To obtain the functional peptide, purification, dissolution, and refolding are necessary.

[0126] Example 2

[0127] This embodiment describes a method for refolding bovine lactoferrin peptide inclusion bodies.

[0128] I. Dissolution of BlfFf 344 inclusion bodies

[0129] 1. Using 10 pre-weighed 50mL centrifuge tubes, dispense 500mL of the fermentation broth obtained in step 2) of Part 3 of Example 1, centrifuge at 6000r / min and 4℃ for 10min, discard the supernatant and collect the bacterial cells.

[0130] 2. Weigh the bacterial cells and resuspend them at a ratio of 0.1g of bacterial cells (wet weight) to 1mL of 1xPBS (NaCl: 137mM, KCl: 2.7mM, Na2HPO4: 10mM, KH2PO4: 2mM, pH=7.2) buffer solution (4℃). Mix the cells slowly by pipetting, then centrifuge at 6000r / min, 4℃ for 10min. Repeat the resuscitation and centrifugation to wash the bacterial cells twice.

[0131] 3. After washing, the bacterial cells were sonicated. The cells were resuspended in 1 mL of 1xPBS buffer (4℃) at a ratio of 0.1 g of bacterial cells, and gently mixed by pipetting. The sonication conditions were: 320 W ultrasonic power, 2 seconds of sonication followed by a 3-second interval, for a total of 20 min. After sonication, the cells were centrifuged at 12000 rpm for 15 min at 4℃ to collect the precipitate.

[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 rpm for 15 min to collect inclusion bodies, repeat the washing and centrifugation twice and remove the supernatant.

[0133] 5. Dissolve the cleaned inclusion bodies in inclusion body dissolving solution (8M urea) at a ratio of 0.1g of bacterial cells to 1mL of inclusion body dissolving solution. Stir the solution with a magnetic stirrer at 4℃ for 2 hours. Then, sonicate the dissolved inclusion body solution until it becomes transparent. The sonication conditions are: 130W ultrasonic power, ultrasonic disruption for 5 seconds, with a 2-second interval, for a total of 2 minutes.

[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, results Figure 3 As shown, where M: molecular weight standard; 1: BlfFf 344 dissolved in 8 M urea.

[0136] II. Purification of peptide BlfFf 344

[0137] 1. In this embodiment, gel filtration chromatography was used to purify the protein. Based on the molecular weight of the protein, a SuperTandex 75 prep grade gel column was selected.

[0138] 2. Column Packing: ① Inspect the chromatography column to ensure all components are intact and clean. Install the lower column head, tighten the O-ring, and fix the column vertically on the stand, ensuring it remains horizontal. ② Add an appropriate amount of packing solution to the column, leaving 1-2 cm of space inside. Continuously pour the solution into the column, avoiding the formation of air bubbles. ③ Connect the upper column head to the low-pressure chromatography system. Start the pump at a certain flow rate to purge any residual air from the tubing. At this point, the medium and solution will separate into layers, and the supernatant will become completely clear. ④ Tighten the O-ring and start the pump at a certain flow rate until the column bed volume no longer changes. Mark the interface. ⑤ Turn off the pump. If there are no more air bubbles and the column volume no longer changes, the column packing is complete.

[0139] 3. Experimental Procedure: ① Equilibration: Before sample loading, equilibrate the chromatography column with 5-10 column volumes of deionized water until the baseline on the recorder becomes stable and the value is 0. ② Sample Loading: Filter the protein solution through a 0.45 μm filter membrane before loading. ③ Elution: Elute with deionized water, maintaining a constant flow rate (1 mL / min) and buffer composition. ④ Collect the protein solution based on the peak value. Figure 4 ).

[0140] Figure 4 The gel filtration chromatography pattern of bovine lactoferrin peptide BlfFf 344 yielded the elution peak (BlfFf). The gel filtration chromatography packing material was SuperTandex G75.

[0141] III. Renaturation of peptide BlfFf 344

[0142] 1. Place the protein solution sample purified by gel filtration chromatography into a dialysis bag for dialysis. In this example, a 14 kDa dialysis bag is used.

[0143] 2. Dialysis bag treatment: ① Cut dialysis bags to an appropriate length based on the volume of the dialysis sample and the unit length of the dialysis bag. ② Immerse the dialysis bag in a container containing 500 mL of dialysis bag treatment solution (2% NaHCO3, 1 mM EDTA, pH 8.0) and place the dialysis bag in a boiling water bath for 10 min. ③ Thoroughly wash the dialysis bag after boiling water bath treatment with distilled water. ④ Place the dialysis bag in a container containing 1 mM EDTA solution and boil it again for 10 min. ⑤ After cooling, transfer it to distilled water or 20% ethanol and store it at 4℃ for later use.

[0144] 3. Place the sample solution into a dialysis bag, and add the dialysis bag to the refolding solution (50mM Tris.Cl, 0.5mM EDTA.Na2, 50mM NaCl, 0.05mM glutathione GSSG, 0.5mM glutathione GSH, 5% glycerol) at a ratio of 1:100. Change the solution with fresh 1xPBS dialysis buffer 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 for 12-24 hours until it becomes 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. Load 10 μL of sample into each well. Run the stacking gel at 80V for 20 min and the separating gel at 120V for 80 min.

[0149] 3. Coomassie brilliant blue staining, observation and photography ( Figure 5 ).

[0150] Figure 5 SDS-PAGE of BlfF 344 after inclusion body dissolution, where M: protein molecular weight standard; 1: dissolved and refolded BlfFf 344 sample (dialysis product obtained in step 3); 2: lyophilized BlfFf 344 sample (10-fold dilution).

[0151] According to testing, in the above process of this embodiment, 1L of fermentation broth yielded a wet weight of 5-8g and a dry weight of 0.5-0.8g of bacterial cells. 1L of fermentation broth can purify 100-150mg of reconstituted bovine lactoferrin lyophilized powder.

[0152] Example 3

[0153] This embodiment tests the antibacterial properties of the refolded polypeptide BlfFf 344 prepared in Example 2.

[0154] 1. Activate *E. coli* DH5α by streaking, and pick single colonies for LB agar culture. Resuspend the bacterial suspension in physiological saline and dilute to 10⁻⁶ CFU / mL. 8 CFU / mL, corresponding bacterial culture OD 600 = Around 0.6.

[0155] 2. Take 50 mL of solid culture medium and heat it until the temperature is suitable. Add a certain amount of DH5α bacterial solution, shake well, and pour it into a petri dish.

[0156] 3. After the culture medium solidifies, holes are punched in the culture medium, and BlfFf344 protein samples are treated with protease, with antibiotics as a positive control and BlfFf344 protein samples not treated with protease as a negative control.

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

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

[0159] Figure 6In the study, a: BlfFf 344 treated with pepsin exhibited antibacterial activity against Escherichia coli; b: 50 μg / mL Amp (ampicillin) solution as control; c: BlfFf 344 protein without pepsin treatment showed no antibacterial activity; d: 10 mg / mL pepsin solution as control. Figure 6 It can be seen that the peptide BlfFf 344 prepared by dissolving and refolding inclusion bodies exhibits complete peptide activity, can be decomposed by pepsin and release antibacterial peptides, and its antibacterial activity is comparable to that of 50 μg / mL ampicillin.

[0160] In summary, this invention induces the expression of the target bovine lactoferrin peptide BlfFf 344 through shake-flask fermentation with *E. coli*. All BlfFf 344 products are insoluble inclusion bodies. This invention leverages the ease of preparation of inclusion bodies; centrifugation allows for the collection of inclusion bodies and the removal of a large amount of impurities. This invention establishes an efficient dissolution process for inclusion bodies. After dissolution, the inclusion bodies are purified by SuperTandex 75 prep grade molecular sieve chromatography. Renaturation conditions are established for the dissolved and purified peptides, enabling the efficient preparation of functionally active BlfFf 344.

[0161] In this 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 for the E. coli expression host is as follows (containing 6XHis):

[0164]

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

[0166] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

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

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 Escherichia coli inducible expression vector into Escherichia coli to obtain genetically engineered recombinant expression Escherichia coli; Using isopropylthiogalactoside to induce the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide BlfFf 344, so that the expression product forms bovine lactoferrin peptide inclusion bodies, thereby obtaining a bacterial cell mixture; The bacterial mixture is centrifuged to collect the Escherichia coli cells, which are then resuspended and subjected to cell wall breaking treatment, followed by centrifugation to collect the bovine lactoferrin peptide inclusion bodies; 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.

2. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, wherein: 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.

3. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, wherein: The method of inducing the genetically engineered recombinant 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 includes: 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 at 25±1°C, and isopropylthiogalactoside is added at a final concentration of 1±0.1 mmol / L to induce efficient expression of the bovine lactoferrin peptide BlfFf 344. The expression product forms bovine lactoferrin peptide inclusion bodies, thereby obtaining a bacterial cell mixture.

4. The method for preparing bovine lactoferrin peptide inclusion bodies according to claim 1, wherein: The method comprises the following steps: centrifuging the bacterial mixture to collect the Escherichia coli cells, resuspending the cells, breaking the bacterial wall, and then centrifuging to collect the bovine lactoferrin peptide inclusion bodies. The bacterial cell mixture is centrifuged, the supernatant is removed, and the Escherichia coli cells are collected; The Escherichia coli cells are resuspended and then subjected to ultrasonic cell wall disruption to obtain an inclusion body mixture; The inclusion body mixture is centrifuged, the supernatant is removed, and the precipitate is collected to obtain bovine lactoferrin peptide inclusion bodies.

5. 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; freeze-drying the bovine lactoferrin peptide renaturation solution 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 4; The amino acid sequence of the bovine lactoferrin peptide BlfFf 344 is shown in SEQ ID NO.

1.

6. The method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 5, 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 an 8.0 mol / L urea solution, stirred and dissolved at 4±1°C for 2.0 h, and then ultrasonically assisted to dissolve until the solution became transparent to obtain a mixed solution; The ratio of the mass of the bovine lactoferrin peptide inclusion bodies to the volume of the urea solution is 1 g:10 mL.

7. The method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 5, 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, and 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.

8. The method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 5, 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 a refolding solution to the bovine lactoferrin peptide solution, and then filling the solution 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%.

9. The method for renaturing bovine lactoferrin peptide inclusion bodies according to claim 8, characterized in that: 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: Adding the refolding solution to the bovine lactoferrin peptide solution, placing the solution into a 14KDa dialysis bag for dialysis at a dialysis temperature of 4±1°C, replacing the dialysate once every 12 hours, and replacing the dialysate 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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