Preparation method of recombinant human lactoferrin based on escherichia coli vector

Abstract

The invention discloses a preparation method of recombinant human lactoferrin based on an escherichia coli vector. The preparation method comprises the following steps: (1) inserting a C leaf segment gene sequence of human lactoferrin into a pET28a plasmid to form a recombinant plasmid pET28a-rhLF; (2) by taking the recombinant plasmid pET28a-rhLF as a template, amplifying to obtain a modified target gene; (3) amplifying by taking the plasmid pET28g as a template to obtain a modified vector; (4) carrying out recombinant connection on the modified target gene and the modified vector, and transferring into DH5alpha to obtain a recombinant plasmid; (5) transferring into Escherichia coli BL21 to obtain an Escherichia coli engineering strain; and (6) carrying out fermentation culture, and carrying out induced expression on the recombinant human lactoferrin. The recombinant human lactoferrin with biological activity can be efficiently expressed in an escherichia coli expression system, the cost is low, the yield is high, and the recombinant human lactoferrin is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly relates to a method for preparing recombinant human lactoferrin based on an Escherichia coli vector. Background Art

[0002] Lactoferrin (LF) is an iron-binding protein with a very high affinity for iron ions, which is 250-300 times that of transferrin. Lactoferrin is an important natural immune protein with various biological activities such as antibacterial, antiviral, antioxidant, and immune regulation. Currently, it has been widely used in cosmetics, food, animal production, medical and other fields as an antioxidant, immune promoter, iron supplement, drug carrier, etc. Direct extraction of lactoferrin from human milk has problems such as limited sources and high costs. Therefore, commercially available lactoferrin is mainly extracted from cow milk. However, there are differences in the amino acid sequence and spatial structure between bovine lactoferrin and human lactoferrin, which may cause immune reactions in the human body and limit its application. Thus, recombinant expression of human lactoferrin has become an important way to solve this problem.

[0003] Currently, common recombinant expression systems include Escherichia coli, yeast, insect cells, mammalian cells, and plant cells. Although the development of plant cell factories and transgenic animal technologies in recent years has made it possible to highly express human lactoferrin in rice or cow milk, these transgenic animal and plant expression systems need to go through multiple stages such as planting, growth, and gene expression, and the whole process takes a long time (several months or even longer). In comparison, the Escherichia coli expression system has significant advantages of fast growth, low cost, and high yield. However, due to the large molecular weight and complex structure of human lactoferrin, its expression level is low in prokaryotic expression systems such as Escherichia coli, and it is difficult to achieve industrial production. In addition, the construction method of a recombinant yeast engineering bacterium OH for soluble expression of recombinant human lactoferrin in CN119776401A mentions that the Escherichia coli expression system lacks a glycosylation modification mechanism, resulting in the inability to produce lactoferrin with biological activity.

[0004] Therefore, how to achieve high-efficiency expression of human lactoferrin in the Escherichia coli expression system and have biological activity has become an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing recombinant human lactoferrin based on an Escherichia coli vector, which can highly express recombinant human lactoferrin with biological activity in the Escherichia coli expression system, has low cost, high yield, and is suitable for industrial production.

[0006] The technical solution adopted by the present invention to solve its technical problems is: A method for preparing recombinant human lactoferrin based on an Escherichia coli vector, comprising the following steps: (1) Recombinant expression was carried out with the C-lobe fragment of human lactoferrin as the target. The gene sequence of the C-lobe fragment of human lactoferrin was inserted into the pET28a plasmid to form the recombinant plasmid pET28a-rhLF; (2) Using the recombinant plasmid pET28a-rhLF as a template, the first modified primer was designed for PCR to obtain the modified target gene; (3) Using the plasmid pET28g as a template, the second modified primer was designed for PCR to obtain the modified vector; (4) The modified target gene obtained in step (2) was recombinantly ligated with the modified vector obtained in step (3). The recombinant ligation product was transferred into DH5α, cultured, extracted to obtain the recombinant plasmid; (5) The recombinant plasmid was transferred into Escherichia coli BL21 to obtain an Escherichia coli engineering strain that highly expresses recombinant human lactoferrin; (6) The Escherichia coli engineering strain that highly expresses recombinant human lactoferrin was fermented and cultured to induce the expression of recombinant human lactoferrin; after the expression product was purified, recombinant human lactoferrin was obtained.

[0007] The present invention selects the C-lobe fragment of human lactoferrin for recombinant expression. This fragment contains the main functional domain of human lactoferrin, has biological activity similar to that of human lactoferrin, and has a relatively small molecular weight. The selected fragment of human lactoferrin in the present invention is special and has functions without glycosylation. The expressed recombinant protein has strong antioxidant biological activity and can be widely applied in the fields of food, medicine, cosmetics, etc.

[0008] To a certain extent, the selection of this fragment in the present invention can also increase the expression level in the Escherichia coli expression system because the molecular weight of the recombinantly expressed protein is small, and the metabolic burden on Escherichia coli will also become smaller. However, this increase is limited. The main reason is that the Escherichia coli plasmid vector was optimized to further increase the expression level.

[0009] The recombinant human lactoferrin expressed in the present invention is different from the natural human lactoferrin sequence, and is expressed by intercepting a partial functional fragment of human lactoferrin.

[0010] The first modified primer was used to perform molecular modification on the base sequence before the start codon of the human lactoferrin C-lobe fragment gene, so that the modified gene fragment could be adapted to the modification site of the modified vector fragment.

[0011] The second modified primer is used to molecularly modify the base sequence between the RBS (ribosome binding site) of the pET vector and the start codon. The modified vector fragment can be ligated with the modified gene fragment to obtain the modified recombinant plasmid. Such modification can improve the expression level of human lactoferrin in Escherichia coli, significantly increase the expression amount of recombinant human lactoferrin in Escherichia coli, and overcome the problem of low expression amount of human lactoferrin in the prokaryotic expression system.

[0012] The gene sequence of the C-lobe fragment of human lactoferrin is shown in SEQ ID NO:2.

[0013] As an embodiment, in step (2), the first modified primer is dT-rhLF F' and rhLF R'; in step (3), the second modified primer is pET28 F' and pET28-dT; The sequence of dT-rhLF F' is shown in SEQ ID NO:3, the sequence of rhLF R' is shown in SEQ ID NO:4, the sequence of pET28 F' is shown in SEQ ID NO:5, and the sequence of pET28-dT is shown in SEQ ID NO:6.

[0014] As another embodiment, in step (2), the first modified primer is dA-rhLF F' and rhLF R'; in step (3), the second modified primer is pET28 F' and pET28-dA R'; The sequence of dA-rhLF F' is shown in SEQ ID NO:7, the sequence of rhLF R' is shown in SEQ ID NO:4, the sequence of pET28 F' is shown in SEQ ID NO:5, and the sequence of pET28-dA R' is shown in SEQ ID NO:8.

[0015] As another embodiment, in step (2), the first modified primer is AT-rhLF F' and rhLF R'; in step (3), the second modified primer is pET28 F' and pET28-AT R'; The sequence of AT-rhLF F' is shown in SEQ ID NO:9, the sequence of rhLF R' is shown in SEQ ID NO:4, the sequence of pET28 F' is shown in SEQ ID NO:5, and the sequence of pET28-AT R' is shown in SEQ ID NO:10.

[0016] The amino acid sequence of recombinant human lactoferrin is shown in SEQ ID NO:1.

[0017] The beneficial effects of the present invention are: it can efficiently express bioactive recombinant human lactoferrin in the Escherichia coli expression system, with low cost and high yield, and is suitable for industrial production. Brief Description of the Drawings

[0018] Figure 1 It is an SDS-PAGE electrophoresis diagram of the expression level of recombinant human lactoferrin in strains induced by IPTG at different temperatures; Figure 2 It is an SDS-PAGE electrophoresis diagram of the expression of recombinant human lactoferrin in different modified strains; Figure 3 It is a comparison diagram of the expression levels of recombinant human lactoferrin in different modified strains; Figure 4 It is an SDS-PAGE electrophoresis diagram of the samples collected at each stage of the nickel ion affinity column in the present invention; Figure 5 It is an SDS-PAGE electrophoresis diagram of the samples collected at each stage of the Q ion column in the present invention; Figure 6 It is a schematic diagram of the appearance (left) and the reconstitution solution (right) of the recombinant human lactoferrin freeze-dried powder obtained in the present invention; Figure 7 It is an SDS-PAGE electrophoresis diagram of the recombinant human lactoferrin obtained in the present invention; Figure 8 It is a verification diagram of the antioxidant activity of recombinant human lactoferrin; Figure 9 It is a plasmid map of pET28a; Figure 10 It is a plasmid map of pET28g. Detailed Embodiments

[0019] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0020] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0021] Example 1: Expression of Recombinant Human Lactoferrin Insert the C-lobe fragment gene sequence of human lactoferrin (SEQ ID NO: 2) into pET28a (i.e., pET - 28a (+), for the plasmid map, see Figure 9)In the plasmid, the recombinant plasmid with correct sequencing results (pET28a-rhLF) was transformed into Escherichia coli BL21. A single colony was picked and inoculated into an LB liquid medium containing kanamycin (50 μg / mL), and cultured overnight in a shaker at 37°C and 220 rpm. The overnight culture broth was taken and inoculated into an LB liquid medium containing kanamycin (50 μg / mL) at a ratio of 1.0%, and cultured in a shaker at 37°C and 220 rpm. When the culture entered the logarithmic growth phase and the OD600 value was measured to be 0.4 - 0.6, IPTG was added to a final concentration of 0.5 mM and induced overnight at 18°C, 25°C, and 37°C respectively. The bacterial liquid was taken for SDS-PAGE electrophoresis to detect the expression level of rhLF (recombinant human lactoferrin).

[0022] As Figure 1 shown, compared with non-induced, obvious specific bands appeared at 39.5 kDa in the strains induced by IPTG at the three temperatures. The expression level was the highest when induced at 37°C. Therefore, 37°C induction was adopted for subsequent screening of high-expression strains.

[0023] Example 2: Vector optimization and screening of high-expression strains Using the pET28a-rhLF plasmid as a template, primers were designed (the modified bases are underlined), as shown in Table 1: Table 1: .

[0024] 2. The plasmid pET28a-rhLF was used as a template, and the primer pairs dT-rhLF F' / rhLF R'; dA-rhLF F' / rhLF R' and AT-rhLF F' / rhLF R' were used to perform PCR to obtain the target gene respectively.

[0025] The PCR systems are shown in Tables 2 - 4 respectively: Table 2 dT-rhLF F' / rhLF R' PCR system: ; Table 3 dA-rhLF F' / rhLF R' PCR system: ; Table 4 AT-rhLF F' / rhLF R' PCR system: .

[0026] PCR program: 1. Pre-denaturation: 98°C, 5 minutes; 2. A total of 32 cycles: Denaturation: 98°C, 15 seconds Annealing: 58 °C, 15 seconds Extension: 72 °C, 20 seconds; 3. Final extension: 72 °C, 5 minutes 4. Incubation: 4 °C, unlimited time.

[0027] Add 10× Loading buffer (loading buffer, TaKaRa) to the PCR product and perform 1% agarose gel electrophoresis. Observe the nucleic acid bands after 150 V, 15 min and cut the gel to recover the target gene.

[0028] 3. Use primer pairs pET28 F' / pET28-dT R', pET28 F' / pET28-dA R' and pET28 F' / pET28-AT R' to perform PCR on pET28g (plasmid map shown in Figure 10 ) to obtain the modified vector.

[0029] In the present invention, the unmodified one in the front can use the pET28a plasmid vector, while the modified one cannot and needs to use another pET28g plasmid vector. Analyzing the reason, it is found that there is an NcoI nuclease cleavage site between the RBS and the start codon ATG in the pET28a plasmid ( Figure 9 shown in the red box), and the recognition sequence of this cleavage site is CCATGG (SEQ ID NO:11). If the NcoI nuclease cleavage site is retained, it will lead to a single-base frameshift mutation. While after the RBS of the pET28g plasmid is the NdeI nuclease cleavage site ( Figure 10 shown in the red box), and the recognition sequence of this cleavage site is CATATG (SEQ ID NO:12), which is the first ATG after the RBS. Changing to the pET28g plasmid will not cause a frameshift mutation.

[0030] The PCR systems are shown in Tables 5 - 7 respectively: Table 5 pET28 F' / pET28-dT R' PCR system: ; Table 6 pET28 F' / pET28-dA R' PCR system: ; Table 7 pET28 F' / pET28-AT R' PCR system: .

[0031] PCR program: 1. Pre-denaturation: 98 °C, 5 minutes; 2. A total of 32 cycles: Denaturation: 98°C, 15 seconds Annealing: 58°C, 15 seconds Extension: 72°C, 1 minute and 40 seconds; 3. Final extension: 72°C, 5 minutes; 4. Incubation: 4°C, infinite time.

[0032] Add 10× Loading buffer (loading buffer, TaKaRa) to the PCR product and perform 1% agarose gel electrophoresis. Observe the nucleic acid bands after 150 V for 15 min and cut the gel to recover the vector.

[0033] 4. Recombinant ligation was performed on the recovered vector and the recovered target gene separately. The recombinant ligation products were separately transformed into DH5α, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, incubated on ice again for 2 - 3 min, added with 500 μL of antibiotic-free LB liquid medium, cultured on a shaker at 37°C for 40 min, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, the precipitate was resuspended with 500 μL of antibiotic-free LB liquid medium, and the resuspended bacterial solution was spread on a Kana plate and cultured overnight in a 37°C incubator. The next day, 5 - 10 monoclonal colonies on the Kana plate (tryptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L, agar 15 g / L, kanamycin 50 μg / mL) were picked and placed into 300 μl of LB liquid medium containing kanamycin (50 μg / mL) respectively, cultured on a shaker at 37°C and 220 rpm for 3 - 4 hours, and the bacterial solution was taken for PCR. Positive colonies were sent for sequencing according to the size of the target band, and strains with complete sequencing results were selected to extract plasmids, namely dA, dT, and AT.

[0034] 5. dA, dT, AT, and the unmodified plasmid (the recombinant plasmid pET28a-rhLF prepared by the method of Example 1) were separately transformed into BL21. Positive BL21 strains were inoculated into 3 mL of LB liquid medium containing kanamycin (50 μg / mL), cultured at 37°C for 4 - 5 h, and then IPTG was added to a final concentration of 0.5 mM and induced overnight at 37°C.

[0035] 6. Take the bacterial solution for SDS-PAGE electrophoresis, analyze the expression level with ImageJ software, and select the strain with the highest expression level. As Figures 2 - 3 shown, compared with the unmodified strain, the dA, dT, and AT modifications can all effectively improve the expression level of rhLF in Escherichia coli, and the AT modification has the most obvious improvement effect. Therefore, the AT strain was selected as the subsequent high-expression fermentation strain.

[0036] Example 3: Purification of recombinant human lactoferrin 1. Add buffer A (50 mM Na2HPO4, 300 mM NaCl, 10% glycerol, 1 mM DTT, pH 7.5) to the cells collected by centrifugation after expression in the AT strain. After thoroughly pipetting to resuspend the cells, homogenize and break the cells 3 - 4 times, centrifuge at 11000 rpm at 4°C for 30 minutes, and retain the pellet. Add buffer B (20 mM Tris, 8 M urea, pH 8.0) to the pellet, stir at room temperature for 1 hour, centrifuge at 11000 rpm at 4°C for 30 minutes, and retain the supernatant. Dilute the supernatant 10 - fold with buffer C (20 mM Tris, 100 mM NaCl), stir evenly, and let it stand overnight at 4°C for renaturation. Replace the solution of the overnight renaturation and concentrate it to buffer D (20 mM Tris, 100 mM NaCl, pH 8.0) to obtain a sample solution containing recombinant human lactoferrin.

[0037] 2. Wash the nickel ion affinity column (Ni - NTA) column material with buffer D, then mix and incubate the column material with the sample solution, gently shake at room temperature or on ice for 30 min, and then load it onto the column. Wash the miscellaneous proteins and elute the proteins with a gradient of buffer D containing 150 mM and 500 mM imidazole. Collect the samples at each stage, perform SDS - PAGE gel electrophoresis for detection. According to the results of the gel pattern ( Figure 4 ), collect the eluate with 150 mM imidazole (with a relatively high content of the target protein), concentrate and replace the solution to buffer E (20 mM Tris - HCl buffer, pH 8.0) and further purify. Wash the Q column (strong anion exchange chromatography column) column material with buffer E, then mix and incubate the column material with the solution of the recombinant lactoferrin after replacing the solution and concentrating, and then load it onto the column. Wash the miscellaneous proteins and elute the proteins with a gradient of buffer E containing 150 mM, 200 mM, 500 mM, and 1 M NaCl. Collect the samples at each stage, perform SDS - PAGE gel electrophoresis for detection. According to the results of the gel pattern ( Figure 5 ), collect the eluates with 150 mM and 200 mM NaCl (with a relatively high purity of the target protein), replace the solution and concentrate it to pure water and then lyophilize.

[0038] As Figure 6 shown, the freeze - dried powder of the recombinant human lactoferrin obtained in the present invention has a reddish - brown appearance. When redissolved at a concentration of 10 mg / mL, the aqueous solution is clear and transparent, and the color is pink. The results of SDS - PAGE electrophoresis show ( Figure 7 ) that the protein purity is about 88%, and the purity is relatively high.

[0039] Example 4: Verification of the antioxidant activity of recombinant human lactoferrin Set up the experimental groups as shown in Table 8: Table 8: .

[0040] The details are as follows: 1. Culture BALB / 3T3 cells in a cell incubator at 37°C and 5% CO2. When the cells grow to 80 - 90% of the culture flask, subculture them. Use complete medium [10% fetal bovine serum + 90% DMEM medium (gibco)], and seed the cells at 5×10 3 cells / well into a 96-well plate. Set three replicate wells for each group and culture them at 37°C and 5% carbon dioxide for 24 hours.

[0041] 2. Discard the supernatant in the wells, and add natural lactoferrin and the recombinant human lactoferrin obtained in Example 3 prepared with 100 μL of maintenance medium [0.4% fetal bovine serum + 99.6% DMEM medium (gibco)] respectively. The final concentrations are both 0.3 mg / mL. Culture them at 37°C and 5% carbon dioxide for 24 hours. The blank control group and the hydrogen peroxide group only add maintenance medium.

[0042] 3. Discard the supernatant in the wells (including the maintenance medium and the test articles), and add hydrogen peroxide prepared with 100 μL of maintenance medium respectively. The final concentration of hydrogen peroxide is 150 μM. Culture them at 37°C and 5% carbon dioxide for 2 hours. The blank control group only adds maintenance medium.

[0043] 4. Discard the supernatant in the wells (including the maintenance medium and hydrogen peroxide), add 100 μL of the reactive oxygen species detection fluorescent probe (Beyotime, catalog number S0033S) prepared with maintenance medium to each well, and culture them at 37°C and 5% carbon dioxide for 20 minutes. Carefully aspirate and discard the supernatant in the wells (including the maintenance medium and the reactive oxygen species detection probe), and wash them 2 - 3 times with maintenance medium. Finally, add 100 μL of maintenance medium to each well, place it in an enzyme - linked immunosorbent assay (ELISA) reader, and use an excitation wavelength of 488 nm and an emission wavelength of 525 nm to detect the fluorescence intensity.

[0044] As Figure 8 shown, compared with natural lactoferrin, the recombinant human lactoferrin of the present invention can also reduce the reactive oxygen species level induced by hydrogen peroxide, and even has a better effect, indicating its strong antioxidant activity.

[0045] The above - described embodiments are only a preferred solution of the present invention, and do not impose any formal restrictions on the present invention. There are other variants and modifications without exceeding the technical solutions described in the claims.

[0046] SEQ ID NO:1: MHHHHHHDDDDKVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK; SEQ ID NO:2:

Claims

1. A method for preparing recombinant human lactoferrin based on an Escherichia coli vector, characterized in that, It includes the following steps: (1) Recombinant expression is carried out with the C-lobe fragment of human lactoferrin as the target, and the gene sequence of the C-lobe fragment of human lactoferrin is inserted into the pET28a plasmid to form the recombinant plasmid pET28a-rhLF; (2) Using the recombinant plasmid pET28a-rhLF as a template, the first modified primer is designed for PCR to obtain the modified target gene; (3) Using the plasmid pET28g as a template, the second modified primer is designed for PCR to obtain the modified vector; (4) The modified target gene obtained in step (2) is recombinantly ligated with the modified vector obtained in step (3), the recombinant ligation product is transferred into DH5α, cultured, extracted, to obtain the recombinant plasmid; (5) The recombinant plasmid is transferred into Escherichia coli BL21 to obtain an Escherichia coli engineering strain that highly expresses recombinant human lactoferrin; (6) The Escherichia coli engineering strain that highly expresses recombinant human lactoferrin is fermented and cultured to induce the expression of recombinant human lactoferrin; after the expression product is purified, recombinant human lactoferrin is obtained.

2. The preparation method according to claim 1, characterized in that, The gene sequence of the C-lobe fragment of human lactoferrin is shown in SEQ ID NO:

2.

3. The preparation method according to claim 1, characterized in that, In step (2), the first modified primer is dT-rhLFF' and rhLF R'; in step (3), the second modified primer is pET28 F' and pET28-dT; The sequence of dT-rhLF F' is shown in SEQ ID NO:3, the sequence of rhLF R' is shown in SEQ ID NO:4, the sequence of pET28 F' is shown in SEQ ID NO:5, and the sequence of pET28-dT is shown in SEQ ID NO:

6.

4. The preparation method according to claim 1, wherein In step (2), the first modified primer is dA-rhLFF' and rhLF R'; in step (3), the second modified primer is pET28 F' and pET28-dA R'; The sequence of dA-rhLF F' is shown in SEQ ID NO:7, the sequence of rhLF R' is shown in SEQ ID NO:4, the sequence of pET28 F' is shown in SEQ ID NO:5, and the sequence of pET28-dA R' is shown in SEQ ID NO:

8.

5. The preparation method according to claim 1, wherein In step (2), the first modified primer is AT-rhLFF' and rhLF R'; in step (3), the second modified primer is pET28 F' and pET28-AT R'; The sequence of AT-rhLF F' is shown in SEQ ID NO:9, the sequence of rhLF R' is shown in SEQ ID NO:4, the sequence of pET28 F' is shown in SEQ ID NO:5, and the sequence of pET28-AT R' is shown in SEQ ID NO:

10.

6. The preparation method according to claim 1, characterized in that, The amino acid sequence of recombinant human lactoferrin is shown in SEQ ID NO:1.

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