Method for expressing human lactoferrin by using lactococcus lactis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a method for expressing human lactoferrin by using lactococcus lactis. The method comprises the following steps: step 1, obtaining a codon-optimized hLF gene nucleotide sequence; 2, constructing a recombinant plasmid containing the hLF gene nucleotide sequence obtained in the step 1; and step 3, transferring the recombinant plasmid obtained in the step 2 into lactococcus lactis for induced expression. The heterologous expression of the human lactoferrin hLF is realized by utilizing the lactococcus lactis, the method is low in cost, convenient and fast, and a new way is provided for high-purity and high-yield preparation of the human lactoferrin hLF.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for expressing human lactoferrin by utilizing Lactococcus lactis. Background Art

[0002] Lactoferrin (LF) is an iron-binding glycoprotein with a molecular weight of approximately 80 kDa. LF is produced by epithelial cells of various organs and is found in a variety of exocrine secretions, including breast milk, tears, nasal and bronchial secretions, saliva, and bile. It belongs to the transferrin family and is capable of binding and transferring iron ions. It is the only protein in the transferrin family that is unaffected by environmental pH, maintaining a consistently high iron-binding activity. The tertiary structure of LF consists of two similar globular lobes, the N lobe and the C lobe. Each lobe is further divided into two subdomains (N1 and N2, and C1 and C2). The cleft between the subdomains can bind one iron ion. LF's most prominent feature is its high affinity for iron, which improves iron bioavailability in intestinal cells, stabilizes reduced iron, and reduces intestinal and gastric irritation. It also possesses antioxidant, antibacterial, and antiviral activities, as well as anticancer and anti-inflammatory properties. LF is also clinically used to improve anemia, treat neonatal sepsis, treat eye diseases, and assist in the treatment of the novel coronavirus.

[0003] Lactoferrin is secreted by a variety of mammals, including humans, cattle, goats, horses, and dogs. Interspecies differences in lactoferrin are minimal, with high similarities in structure and function. Human lactoferrin (hLF) is a non-heme iron-binding protein composed of 692 amino acids and a molecular weight of approximately 80 kDa. hLF shares 70% sequence identity with bovine lactoferrin (bLF) and ovine lactoferrin (oLF), and their tertiary structures are also highly similar. Despite this, hLF exhibits significant differences from LF from other sources, potentially offering advantages for its use in food or pharmaceutical applications. Studies have shown that hLF specifically binds to receptors on intestinal brush-border membrane vesicles. Both half-LF and deglycosylated LF bind to the receptors with similar affinity to intact LF. However, bovine lactoferrin exhibits minimal binding to intestinal brush-border membrane vesicles, suggesting that human lactoferrin may have a higher affinity for some human receptors. Wang et al. compared the effects of hLF, bLF, and recombinant human lactoferrin (rhLF) on Caco-2 cell line function (cell proliferation, differentiation, and barrier function establishment) and found that rhLF and hLF had more similar effects than bLF and hLF. Therefore, rhLF may be a better choice for infant formula compared to bLF.

[0004] Currently, LF is primarily purified from milk or colostrum using methods such as salting-out, chromatography, membrane separation, and magnetic nanoparticles. However, the low concentration of LF in milk results in low yields and high extraction costs. In July 2017, China implemented the new national food safety standard, "Lactoferrin, a Food Nutrient Fortifier" (GB 1903.17-2016), which revised the physical and chemical specifications of LF and raised the purity requirement to 95%. This placed higher demands on LF production technology, resulting in a significant market gap for LF, with supply exceeding demand and prices soaring. Over the past decade, researchers have explored the production of LF using genetic engineering techniques. Compared to other expression systems, bacterial expression of LF offers the advantages of simplicity and low cost. Numerous studies have successfully expressed LF in bacteria such as Escherichia coli and Lactobacillus casei. In summary, biosynthesis (i.e., heterologous expression) is a viable approach for obtaining large quantities of high-purity, active LF.

[0005] Lactococcus lactis is a gram-positive bacterium, which is a generally recognized as safe (GRAS) microorganism and is commonly used in food fermentation, drug production, etc. Lactococcus lactis has the characteristics of fast growth, low cost, easy operation, clear genetic background, etc. Therefore, researchers are constantly developing it as a host for the overexpression of homologous or heterologous proteins. With the help of genetic engineering and biotechnology, a series of cloning and expression systems with Lactococcus lactis as the core have been established and developed. The Lactococcus lactis expression system can be divided into constitutive expression system and inducible expression system. The gene expression of the constitutive system is not affected by the growth period, environment, etc., and the gene expression of the inducible system is stimulated by specific signals to show different expression states. Among them, the nisin-controlled expression (NICE) system is the most successful in Lactococcus lactis, and it is also the only commercial expression system in Lactococcus lactis. The expression of the gene is induced by adding sub-inhibitory concentration (0.1-5 ng / mL) of nisin to the culture medium. When the membrane protein NisK senses nisin, it undergoes autophosphorylation and transfers the phosphate group to activate the response regulator protein NisR, which in turn induces the promoter PnisA to start transcription and express the target gene. The host strains of the NICE system are all derived from L. lactis subsp. Cremoris MG1363 (the plasmid-free descendant of dairy fermentation strain NCDO712), and the commonly used ones are NZ9700, NZ9800, NZ9000, NZ3900 and NZ3000, etc. Using Lactococcus lactis as a host for expressing heterologous proteins has the following advantages: (1) Lactococcus lactis is easy to culture, easy to transfer foreign genes, and low in cost; (2) Lactococcus lactis is a food-grade strain, which is safe and does not produce endotoxin, and the protein expressed by it can be directly taken orally; (3) Lactococcus lactis secretes less extracellular proteases, reducing the probability of degradation of secreted proteins; (4) Lactococcus lactis secretes less proteins, reducing the difficulty of isolating target proteins; (5) Lactococcus lactis is not sensitive to the inhibitory effect of lactoferrin. Based on the above advantages, the Lactococcus lactis expression system is widely used in food and pharmaceutical industries, such as production of proteins and peptides, antigen presentation, etc.

[0006] However, there is currently no report on using Lactococcus lactis as an expression host to produce human lactoferrin. SUMMARY

[0007] To solve the above problems, the present application uses Lactococcus lactis as an expression host and attempts to express recombinant hLF by heterologous expression, in order to provide a low-cost and convenient method for the production of lactoferrin.

[0008] The present invention provides a method for expressing human lactoferrin using Lactococcus lactis, the method comprising the following steps:

[0009] Step 1: obtaining a codon-optimized hLF gene nucleotide sequence;

[0010] Step 2: constructing a recombinant plasmid containing the hLF gene nucleotide sequence obtained in step 1;

[0011] Step 3: The recombinant plasmid obtained in step 2 is transferred into Lactococcus lactis for inducing expression.

[0012] In one embodiment of the present invention, the codon-optimized hLF gene nucleotide sequence is shown as SEQ ID NO.4.

[0013] In one embodiment of the present invention, the recombinant plasmid is pNZ8148-hLF.

[0014] In one embodiment of the present invention, the Lactococcus lactis is selected from one of NZ9700, NZ9800, NZ9000, NZ3900 or NZ3000.

[0015] In one embodiment of the present invention, the Lactococcus lactis is NZ9000.

[0016] In one embodiment of the present invention, the transfer method in step 3 is electroporation.

[0017] In one embodiment of the present invention, the step of inducing expression in step 3 is to transfer the Lactococcus lactis containing the recombinant plasmid into the fermentation medium at an inoculation rate of 4-7%, preferably 5%, for fermentation culture.

[0018] In one embodiment of the present invention, the fermentation culture conditions are: temperature 28-32°C, time 14-18 hours. Preferably, the temperature is 28°C, 29°C, 30°C, 31°C, 32°C, and the time is 14 hours, 15 hours, 16 hours, 17 hours, 18 hours.

[0019] Compared to the prior art, the present invention constructs a lactic acid bacteria expression vector pNZ8148-hLF containing the full-length hLF gene sequence. This vector is then electroporated into Lactococcus lactis NZ9000 and screened for positive recombinant bacteria. After induction, the recombinant Lactococcus lactis demonstrates specific expression of the target protein using SDS-PAGE. This method provides a low-cost, convenient, and rapid heterologous expression method for human lactoferrin hLF, offering a new approach for producing high-purity and high-yield human lactoferrin hLF. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The cells are human mammary epithelial cells MCF-10a.

[0021] Figure 2 The electrophoresis diagram of total RNA from human mammary epithelial cells MCF-10a; lanes 1-3 are RNA samples from MCF-10a.

[0022] Figure 3 Agarose gel electrophoresis of PCR products.

[0023] Figure 4 This is the blue-white screening of the hLF gene after TA cloning.

[0024] Figure 5 Agarose gel electrophoresis diagram for PCR verification of positive clone colonies. Lanes 1-10 represent 10 picked positive clone colonies.

[0025] Figure 6 Agarose gel electrophoresis diagram of the double enzyme digestion product.

[0026] Figure 7 This is the map of the recombinant plasmid pNZ8148-hLF.

[0027] Figure 8 The figure is agarose gel electrophoresis diagram of PCR verification of positive clones.

[0028] Figure 9 The figure shows the SDS-PAGE analysis of the expression products.

[0029] Figure 10 This is the change curve of OD value and culture medium pH during 24 hours of fermentation of recombinant Lactococcus lactis NZ9000. DETAILED DESCRIPTION

[0030] The following describes the specific embodiments of the present invention with reference to the accompanying drawings. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0031] Human mammary epithelial cell complete culture medium: DMEM + 5% horse serum + 20 ng / mL epidermal growth factor + 0.5 μg / mL hydrocortisone + 10 μg / mL insulin + 1% non-essential amino acids + 1% P / S.

[0032] Example 1 Culture of human mammary epithelial cells MCF-10a

[0033] 1. Cell Thawing: Before the experiment begins, add ultrapure water to a 1L beaker and heat the beaker in a 37°C water bath. In a cleanroom, add 10mL of complete culture medium to a new culture flask and place it in a cell culture incubator to equilibrate the pH and temperature for 15 minutes. Remove the cryovial from the liquid nitrogen tank and immediately place it in 37°C water to completely thaw the frozen cell suspension. Disinfect the cryovial by spraying it with 75% alcohol and then transfer it to the cleanroom. Transfer the cell suspension to a 15mL centrifuge tube. Centrifuge the tube at 1000g for 3 minutes to pellet the cells. Discard the supernatant and add 3mL of fresh complete culture medium. Gently pipette to resuspend the cells. Transfer the cell suspension to a culture flask and incubate the flask in a 37°C incubator with 5% CO2 for 12 hours. Once the cells have adhered, replace with fresh complete culture medium.

[0034] 2. Cell Passaging: Observe under a microscope. Passage cells when 70%-90% of the cells within the field of view are adherent. Remove the culture medium, PBS solution, and trypsin from the 4°C refrigerator and equilibrate to room temperature. Remove the culture flask from the incubator, spray the flask mouth with 75% alcohol to disinfect, and then bring it to the clean bench for processing. Use a 10mL pipette to aspirate the old culture medium and discard. Wash the cells twice with 5mL of PBS. Add 1.5mL of trypsin to the flask, gently shake the flask to ensure the trypsin covers the entire bottom of the flask, and incubate in a 37°C incubator for 10 minutes to digest. Observe the cells under an inverted microscope. Digestion is complete when the cells become rounded, the spaces between cells become larger, and the cell membrane becomes transparent. Return the flask to the clean bench and add 4mL of fresh culture medium to terminate digestion. Repeatedly pipette the cells against the flask wall, being careful to gently remove the cells and form a cell suspension. Use a long pipette to transfer the cell suspension into a 15mL centrifuge tube and centrifuge at 1000g for 3 minutes. After centrifugation, discard the supernatant and resuspend the cells in 5-10 mL of fresh complete medium in a T25 culture flask. Gently pipette to mix thoroughly, then add the cells to the culture flasks in a 1:2 ratio. Add fresh complete medium and continue culturing in a 5% CO2, 37°C incubator. Subculture cells every two days.

[0035] 3. Cell Cryopreservation: When cells have grown to 80%-90% adherent monolayer in the culture flask, they can be cryopreserved at a 1:4 ratio. First, remove and discard the old culture medium from the culture flask. Wash the cells twice with 5 mL of PBS, then add 15 mL of trypsin and digest at 37°C for 10 minutes. After digestion, add 4 mL of fresh culture medium to terminate the digestion. Use a long disposable pipette to repeatedly pipette the cells to form a cell suspension. Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 g for 3 minutes. Prepare freezing solution at a ratio of 9:1 horse serum to DMSO, mix thoroughly, and set aside. After centrifugation, discard the supernatant in the tube and add an appropriate amount of freezing solution to the tube. Gently pipette to resuspend the cells, then add 1 mL of cell suspension to each cryovial. Label the cryovials, seal them with parafilm, and place them in a 4°C refrigerator for 30 minutes. Then, transfer them to a -20°C refrigerator for 30 minutes, then to a -80°C refrigerator overnight. Transfer them to a liquid nitrogen tank for long-term storage.

[0036] Example 2 hLF gene amplification

[0037] 1. Total RNA extraction from human mammary epithelial cells MCF-10a

[0038] 1) Human mammary epithelial cells MCF-10a were lysed by adding lysis buffer to a 6-well culture plate. 6 Add 1 mL of lysis buffer to the cells and mix thoroughly by pipetting with a sampler.

[0039] 2) The treated sample was placed at room temperature for 5 minutes to allow the nucleic acid-protein complex to be completely separated.

[0040] 3) Add 0.2 mL of chloroform to the homogenate sample, cap the tube, shake vigorously for 15 seconds, and let it stand at room temperature for 3-5 minutes.

[0041] 4) Centrifuge at 12,000 rpm for 10 minutes at 2-8°C. The RNA is primarily in the colorless upper aqueous phase. Transfer the aqueous phase to a fresh tube, avoiding the pellet.

[0042] 5) Add 500 μL of washing solution to the adsorption column, incubate at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes at 2-8°C, and discard the waste liquid.

[0043] 6) Add 200 μL of anhydrous ethanol to the supernatant collected in step 4, mix well, add to the adsorption column, let it stand for 2 minutes, centrifuge at 2-8°C and 12000 rpm for 2 minutes, and discard the waste liquid.

[0044] 7) Add 600 μL of rinse solution to the column, centrifuge at 12,000 rpm for 2 minutes at 2-8°C, and discard the waste solution. Repeat this step once.

[0045] 8) Centrifuge at 12000 rpm for 2 minutes, discard the collection tube, and place the adsorption column at room temperature for several minutes to remove the remaining rinse solution in the adsorption column.

[0046] 9) Place the adsorption column in a new tube, add 50-100 μl of RNase-free ddH2O to the center of the membrane, let it stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 2 minutes at room temperature to obtain RNA.

[0047] 2. cDNA Synthesis

[0048] Total RNA was transcribed into cDNA according to the instructions of the Solebro Universal Reverse Transcription Kit.

[0049] 1) Add the reaction components listed in Table 1 to an RNase-free centrifuge tube on ice.

[0050] Table 1 Reaction components

[0051] Element Dosage Total RNA 5 μg Oligo(dT) 16 ]] 2μL <![CDATA[RNase-free ddH2O]]> Up to 14.5μL

[0052] 2) After incubation at 70°C for 5 minutes, quickly cool on ice for 2 minutes. After brief centrifugation, collect the reaction solution and add the following components.

[0053] Table 2 Reaction components

[0054] Element Dosage 5×M-MLV Buffer 4 μL dNTPs 1 μL RNasin 0.5μL M-MLV 1 μL

[0055] 3) Warm bath at 42°C for 60 minutes.

[0056] 4) Terminate the reaction by heating at 95°C for 5 min and place on ice for subsequent experiments.

[0057] 3. hLF fragment DNA amplification

[0058] 1) Primer design and synthesis: Primers were designed based on the conserved sequence of lactoferrin: hLF-F: 5'-atgaaacttgtcttcctcgtcc-3', hLF-R: 5'-ttacttcctgaggaattcacagg-3'.

[0059] 2) Add the reaction components shown in Table 3 into the EP tube.

[0060] Table 3 Amplification reaction components

[0061] Element Dosage 2×Taq Master Mix 25 μL hLF-F (10 μM) 2μL hLF-R (10 μM) 2μL cDNA 1 μL <![CDATA[ddH2O]]> Up to 50μL

[0062] 3) PCR reaction conditions were as follows: initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 2 min 30 s; and a final extension at 72°C for 5 min. After amplification, PCR results were analyzed by 1% agarose gel electrophoresis.

[0063] Example 3 Subcloning and identification of hLF gene

[0064] 1. Subcloning of hLF gene

[0065] After agarose gel electrophoresis, excise the single target DNA band from the agarose gel, place it in a clean centrifuge tube, and weigh it. Recover the hLF DNA band according to the Solebro Gel Extraction Kit instructions. Next, ligate the hLF DNA sample to the pMD-18T vector using the ligation system shown in Table 4. Add 5 μL of Solution 1 and incubate at 16°C for 6 hours. Then, transform the recombinant plasmid into competent E. coli JM109 cells.

[0066] Table 4 Connection system

[0067] Element Dosage pMD18-T Vector 1 μL hLF DNA 2μL sterile water Up to 5μL

[0068] 2. Identification of recombinant plasmid pMD-18T-hLF

[0069] 1) Add the PCR validation components shown in Table 5 to a microcentrifuge tube.

[0070] Table 5 PCR verification components

[0071] Element Dosage hLF-F (10 μM) 0.5μL hLF-R (10 μM) 0.5μL 2×Taq PCR MasterMix 12.5μL <![CDATA[ddH2O]]> Up to 25μL

[0072] 2) Use a toothpick to pick up the positive clones on the screening plate, place them in a microcentrifuge tube, and stir several times.

[0073] 3) PCR reaction conditions were as follows: initial denaturation at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes and 30 seconds; and a final extension at 72°C for 8 minutes. After amplification, PCR results were analyzed by 1% agarose gel electrophoresis. The PCR product was sequenced and compared with the published human lactoferrin gene sequence at NCBI.

[0074] Example 4 Codon Optimization

[0075] Based on the codon usage preference of Lactococcus lactis NZ9000, a variant hLF was constructed to improve protein production. The codon-optimized hLF gene sequence was synthesized by Biomed, and then hLF was ligated with the pUC57 vector to construct the recombinant expression plasmid pUC57-hLF, which was then transformed into E. coli.

[0076] Example 5 Construction and identification of recombinant expression system

[0077] 1. Construction of the recombinant expression system pNZ8148

[0078] Plasmid extraction: The pMD-18T plasmid containing the hLF gene was extracted from E. coli according to the instructions of the Solebro Plasmid Miniprep Kit.

[0079] Double enzyme digestion: The plasmid extracted above was double digested with Sac I and Kpn I. The enzyme digestion reaction system is shown in Table 6.

[0080] Table 6 Double enzyme digestion system

[0081] Element Dosage 10×NEB Buffer 2μL 100×BSA 0.2μL Kpn I 0.5μL Sac I 0.5μL Recombinant plasmid DNA / pNZ8148 empty plasmid 6.8μL <![CDATA[ddH2O]]> Up to 25μL

[0082] Gently mix the above components and incubate in a 37°C water bath for 5 hours. Observe 5 μL of the digestion product by 1% agarose gel electrophoresis. Excise the hLF gene fragment and the pNZ8148 empty plasmid fragment and recover the digestion products according to the DNA gel recovery kit instructions. Store the recovered hLF gene and pNZ8148 empty plasmid fragments at -20°C until further use.

[0083] Preparation of competent Lactococcus lactis NZ9000:

[0084] 1) The preserved lactic acid bacteria NZ9000 strain was aseptically inoculated into 5 mL of GM17 liquid culture medium and cultured at 30°C for 24 h.

[0085] 2) Inoculate 500 μL of the culture into 10 mL of GM17 liquid medium and continue to culture at 30°C for 24 h.

[0086] 3) Inoculate 10 mL of the culture into 100 mL of GM17 liquid medium and continue culturing for approximately 3-4 hours until the bacteria enter the logarithmic growth phase (OD600 = 0.4).

[0087] 4) Transfer the bacteria into two sterile 50 mL centrifuge tubes pre-cooled on ice and place them on ice for 10 minutes to cool the culture to 0°C.

[0088] 5) Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, recover the bacterial pellet, add 50 mL of pre-chilled 0.5 M sucrose / 10% glycerol to each tube to resuspend the pellet, and let it rest on ice for 20 min.

[0089] 6) Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, recover the bacterial pellet, add 25 mL of pre-chilled 0.5 M sucrose / 10% glycerol / 0.05 M EDTA to each tube to resuspend the pellet, and let it rest on ice for 20 min.

[0090] 7) Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, recover the bacterial pellet, add 25 mL of pre-chilled 0.5 M sucrose / 10% glycerol to each tube to resuspend the pellet, and let it rest on ice for 20 min.

[0091] 8) Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, recover the bacterial pellet, add 500 μL of pre-chilled 0.5 M sucrose / 10% glycerol to each tube to resuspend the pellet, aliquot 40 μL per tube, and store at -80°C until needed.

[0092] Enzyme ligation: Ligate the target fragment and expression plasmid pNZ8148. The ligation reaction system is shown in Table 7. Ligation was carried out at 16°C overnight.

[0093] Table 7 Enzyme-linked reaction system

[0094] Element Dosage Recovered hLF fragment 12 μL Recovered pNZ8148 plasmid fragment 4 μL T4 DNA Ligase 2μL 10×Ligase Buffer 2μL

[0095] Transformation: Transform the recombinant plasmid into lactic acid bacteria NZ9000: Remove competent cells from a -80°C freezer, thaw on ice, add 1 μL of the purified ligation product, place in a 2 mm pre-chilled electroporation cuvette, and perform electroporation. Electroporation parameters: 2 kV, 4.5 ms. After electroporation, add 1 mL of pre-chilled GM17 recovery medium and transfer the culture to a 1.5 mL centrifuge tube. Incubate on ice for 5 minutes and then at 30°C for 2 hours. Spread 20 μL of the culture onto selection medium containing chloramphenicol and incubate at 30°C for 24 hours. Observe for the presence of colonies.

[0096] 2. Identification of recombinant plasmid pNZ8148-hLF

[0097] The PCR verification method and sequencing verification method refer to Example 3.

[0098] Example 6 Expression and Identification of Recombinant Lactococcus lactis hLF Gene

[0099] 1. Inducible expression of recombinant Lactococcus lactis hLF gene

[0100] 1) Using empty vector Lactococcus lactis NZ9000 as a control, Lactococcus lactis containing the recombinant plasmid and the control group were inoculated into GM17 liquid culture medium at a ratio of 1:100 and cultured at 30°C for 24 h.

[0101] 2) The culture was inoculated into 5 mL of GM17 liquid medium at a ratio of 1:25 and cultured for about 3-4 hours until the bacteria entered the logarithmic growth phase (OD600 = 0.4).

[0102] 3) Induce with 10 ng / mL Nisin for 3-4 hours and then terminate the culture.

[0103] 2. Analysis of expression products by SDS-polyacrylic acid gel electrophoresis (SDS-PAGE)

[0104] 1) Adjust the OD600 of the bacterial solution harvested after induction to 0.5.

[0105] 2) Take 1.5 mL of bacterial solution and centrifuge at 12,000 rpm at 4°C for 3 min. Discard the supernatant. Wash the pellet with 200 μL of PBS solution and centrifuge at 12,000 rpm at 4°C for 3 min. Discard the supernatant.

[0106] 3) Add 100 μL of 10 mg / mL lysozyme, place in a 37°C water bath for 30 min, and then boil in water for 5 min to inactivate the lysozyme.

[0107] 4) Centrifuge at 12000 rpm at 4°C for 3 min, discard the supernatant, add 25 μL of TE and an appropriate amount of 5× SDS gel loading buffer (containing 0.2 mol / L DTT), mix, and boil in boiling water for 10 min.

[0108] 5) The PAGE gel of SDS-PAGE experiment was prepared according to the TM Prepared according to the instructions of the one-step PAGE gel rapid preparation kit.

[0109] 6) Sample loading: Inject electrophoresis buffer into the electrophoresis tank, take 10 μL of the treated sample supernatant and load it, using 10 kDa to 250 kDa as the standard.

[0110] 7) Electrophoresis: Perform electrophoresis at a constant voltage of 80 V. After the sample enters the separating gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue is 0.5 cm from the bottom edge of the glass plate.

[0111] 8) Staining: Soak the gel in at least 5 volumes of Coomassie Brilliant Blue staining solution and stain on a horizontal shaker for about 2 hours.

[0112] 9) Destaining: Soak the gel in destaining solution and destain on a horizontal shaker, changing the destaining solution during destaining, until the background is clear.

[0113] 10) Glue.

[0114] Example 7 Small-scale fermentation of recombinant Lactococcus lactis

[0115] Activated Lactococcus lactis containing the pNZ8148 empty plasmid and the pNZ8148-hLF recombinant plasmid were transferred to a fermenter containing 3 L of fermentation medium at a 5% inoculum size. The fermenter was stirred at 100 rpm, aerated at 0.9 L / min, and incubated at 30°C for 16 hours. The OD600 and pH of the culture medium were measured every 2 hours.

[0116] Effect Examples

[0117] Effect Example 1 Amplification of hLF gene

[0118] like Figure 1Total RNA was extracted from human mammary epithelial MCF-10a cells as shown in the figure and immediately reverse-transcribed into cDNA. The extracted RNA and cDNA concentrations were 297.10 ng / μL and 807.21 ng / μL, respectively. Figure 2 This is the electrophoresis diagram of total RNA from human mammary epithelial cells MCF-10a. The two bright bands are 5s and 18s, while 25s may be degraded into a fragment of 1500bp.

[0119] At the same time, primers hLF-F: 5'-atgaaacttgtcttcctcgtcc-3' (SEQ ID NO. 1) and hLF-R: 5'-ttacttcctgaggaattcacagg-3' (SEQ ID NO. 2) were designed based on the conserved region sequence of lactoferrin to amplify the hLF gene. After verification by agarose gel electrophoresis, a clear single band was obtained around 2100 bp, and the nucleotide fragment size was consistent with the expected result. Figure 3 shown.

[0120] Effect Example 2 Construction of Subcloning Vector

[0121] The band obtained above was recovered by gel extraction, connected with plasmid pMD18-T, and the plasmid was transformed into E. coli JM109. The bacterial solution was spread on LB medium containing Amp, IPTG, and X-gal ( Figure 4 ). 10 white colonies were picked and verified by PCR using hLF gene primers. Except for colonies 7 and 10, all others were positive clone colonies ( Figure 5 ).

[0122] Effect Example 3 hLF DNA Sequence Verification

[0123] The hLF gene linked to the recombinant plasmid pMD18-T-hLF was sequenced and verified, resulting in a complete sequence (SEQ ID NO. 3). Nucleic acid comparison with the corresponding hLF gene sequence in NCBI (GenBank: U07643.1) revealed a 99.9% homology to the mRNA sequence of the hLF gene submitted by other researchers, indicating successful cloning of the hLF gene.

[0124] Effect Example 4 Codon Optimization

[0125] According to the codon usage preference of Lactococcus lactis, the obtained hLF sequence was codon optimized to obtain the optimized sequence (SEQ ID NO.4) and two restriction enzyme sites, Kpn I and Sac I, were added at both ends of the optimized sequence, respectively.

[0126] Effect Example 5: Construction of Gene Expression Vector in Lactococcus lactis

[0127] The recombinant plasmid containing hLF and the expression plasmid pNZ8148 of Lactococcus lactis NZ9000 were double-digested with Kpn I and Sac I ( Figure 6 ). The recombinant plasmid containing hLF was digested to obtain two bands of 2100 bp and 2700 bp, respectively, which are the hLF band and the plasmid band. The pNZ8148 plasmid was digested to obtain a band of approximately 3100 bp.

[0128] The target band was recovered by gel extraction, and the hLF fragment was ligated with plasmid pNZ8148 to construct the recombinant plasmid pNZ8148-hLF. The recombinant plasmid map is shown in Figure 2. Figure 7 shown.

[0129] Effect Example 6 Construction of recombinant Lactococcus lactis

[0130] The recombinant plasmid pNZ8148-hLF was electroporated into Lactococcus lactis NZ9000, and positive clones were picked from the screening plate for PCR verification. Among the 39 single colonies picked, 38 positive clones were found ( Figure 8 Select the positive clone colonies with higher band brightness for subsequent experiments ( Figure 8 (sample in lane 34).

[0131] Effect Example 7 Induced Expression of Recombinant Lactococcus lactis hLF Gene

[0132] The NZ9000 strain containing the recombinant plasmid pNZ8148-hLF and the empty plasmid strain were induced. The pNZ8149-NZ3900 empty plasmid strain did not express the target size protein, while the NZ9000 strain containing the recombinant plasmid pNZ8149-hLF expressed a protein with a molecular weight of approximately 80 kDa, indicating that the recombinant human lactoferrin was expressed in a soluble form in the cells ( Figure 9 ).

[0133] Effect Example 8: Fermentation test of recombinant Lactococcus lactis NZ9000

[0134] The recombinant Lactococcus lactis NZ9000 and the original Lactococcus lactis NZ9000 strains were used to conduct a small-scale experiment in a 5L fermentation tank. The fermentation process was monitored, and the changing trends of OD600 and pH during the fermentation process showed that ( Figure 10 ), the fermentation characteristics of the recombinant strain were similar to those of the original strain, which proved that carrying the plasmid and nisin induction would not have a significant impact on the fermentation of the strain.

[0135] In conclusion, the hLF gene is successfully cloned, and the hLF gene lactobacillus expression vector pNZ8148-hLF is constructed through PCR and DNA sequencing identification after T-A cloning.

[0136] Although the present application has been disclosed with reference to preferred embodiments, it is not intended to limit the present application thereto, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for expressing human lactoferrin using Lactococcus lactis, characterized in that: The method The following steps are included: Step 1, obtaining a codon-optimized hLF gene nucleotide sequence; Step 2: constructing a recombinant plasmid containing the hLF gene nucleotide sequence obtained in step 1; Step 3: The recombinant plasmid obtained in step 2 is transferred into Lactococcus lactis for inducing expression.

2. The method according to claim 1, characterized in that The codon-optimized hLF gene nucleotide sequence is shown in SEQ ID NO.

4.

3. The method according to claim 1, characterized in that The recombinant plasmid is pNZ8148-hLF.

4. The method according to claim 1, wherein The Lactococcus lactis is selected from one of NZ9700, NZ9800, NZ9000, NZ3900 or NZ3000.

5. The method according to claim 1, wherein The Lactococcus lactis is NZ9000.

6. The method according to claim 1, characterized in that The transfer method in step 3 is electroporation.

7. The method according to claim 1, characterized in that The step of inducing expression in step 3 is to transfer the Lactococcus lactis containing the recombinant plasmid into the fermentation medium at an inoculation rate of 4-7% for fermentation culture.

8. The method according to claim 7, characterized in that The fermentation culture conditions are as follows: temperature 28-32° C., and culture 14-16 hours.

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