A preparation method of recombinant human lactoferrin based on Escherichia coli vector

Abstract

The present invention discloses a method for preparing recombinant human lactoferrin based on an Escherichia coli vector, comprising the following steps: (1) inserting the C lobe fragment gene sequence of human lactoferrin into a pET28a plasmid to form a recombinant plasmid pET28a-rhLF; (2) using the recombinant plasmid pET28a-rhLF as a template, amplifying to obtain a modified target gene; (3) using the plasmid pET28g as a template, amplifying to obtain a modified vector; (4) recombinantly connecting the modified target gene with the modified vector, and transferring them into DH5α to obtain a recombinant plasmid; (5) transferring them into Escherichia coli BL21 to obtain an Escherichia coli engineered strain; and (6) fermenting and culturing to induce expression of recombinant human lactoferrin. The present invention can efficiently express recombinant human lactoferrin with biological activity in an Escherichia coli expression system, has low cost and high yield, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular 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 high affinity for iron ions, 250-300 times that of transferrin. Lactoferrin is an important natural immune protein with numerous biological activities, including antibacterial, antiviral, antioxidant, and immune-regulating activities. It is currently used as an antioxidant, immunostimulant, iron supplement, and drug carrier in cosmetics, food, animal production, and healthcare. Direct extraction of lactoferrin from human milk is limited in availability and expensive, so commercially available lactoferrin is primarily extracted from bovine milk. However, differences in amino acid sequence and spatial structure between bovine and human lactoferrin can lead to immune responses in the human body, limiting its application. Therefore, recombinant expression of human lactoferrin has become an important approach to address this issue.

[0003] Currently, common recombinant expression systems include Escherichia coli, yeast, insect cells, mammalian cells, and plant cells. Although recent developments in plant cell factories and transgenic animal technology have enabled efficient expression of human lactoferrin in rice or cow milk, these transgenic plant and animal expression systems require multiple stages, including planting, growth, and gene expression, making the entire process time-consuming (several months or even longer). In comparison, E. coli expression systems offer significant advantages such as rapid growth, low cost, and high yield. However, due to the large molecular weight and complex structure of human lactoferrin, its expression levels in prokaryotic expression systems such as E. coli are low, making industrial production difficult. Furthermore, CN119776401A, a method for constructing a recombinant yeast strain OH that solublely expresses recombinant human lactoferrin, states that the E. coli expression system lacks a glycosylation modification mechanism, resulting in the inability to produce biologically active lactoferrin.

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

[0005] The present invention aims to provide a method for preparing recombinant human lactoferrin based on an Escherichia coli vector, which can efficiently express recombinant human lactoferrin with biological activity in an 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 problem is:

[0007] A method for preparing recombinant human lactoferrin based on an Escherichia coli vector comprises the following steps:

[0008] (1) The C lobe fragment of human lactoferrin was used as the target for recombinant expression, and the gene sequence of the C lobe fragment of human lactoferrin was inserted into the pET28a plasmid to form the recombinant plasmid pET28a-rhLF;

[0009] (2) Using the recombinant plasmid pET28a-rhLF as a template, the first modified primer was designed for PCR to obtain the modified target gene;

[0010] (3) Using plasmid pET28g as a template, design the second modified primer for PCR to obtain the modified vector;

[0011] (4) Recombinantly connecting the modified target gene obtained in step (2) with the modified vector obtained in step (3), transferring the recombinant connection product into DH5α, culturing, extracting, and obtaining a recombinant plasmid;

[0012] (5) The recombinant plasmid was transformed into Escherichia coli BL21 to obtain an Escherichia coli engineered strain that efficiently expressed recombinant human lactoferrin;

[0013] (6) The engineered Escherichia coli strain that efficiently expresses recombinant human lactoferrin is fermented and cultured to induce the expression of recombinant human lactoferrin; the expression product is purified to obtain recombinant human lactoferrin.

[0014] The present invention selects the C lobe fragment of human lactoferrin for recombinant expression. This fragment contains the main functional domains of human lactoferrin and has similar biological activity to human lactoferrin, but with a smaller molecular weight. The human lactoferrin fragment selected in the present invention is unique and does not require glycosylation to function. The expressed recombinant protein has strong antioxidant bioactivity and can be widely used in the fields of food, medicine, cosmetics, etc.

[0015] The selection of this fragment in the present invention can also improve expression in E. coli expression systems to a certain extent, because the recombinantly expressed protein has a small molecular weight, which also reduces the metabolic burden on E. coli. However, this improvement is limited, mainly due to the optimization of the E. coli plasmid vector, which further improves expression.

[0016] The recombinant human lactoferrin expressed by the present invention is different from the natural human lactoferrin sequence, and is expressed by intercepting a partial functional fragment of human lactoferrin.

[0017] The first modified primer is used to carry out molecular modification on the base sequence before the start codon of the human lactoferrin C lobe fragment gene, so that the modified gene fragment can be compatible with the modified site of the modified vector fragment.

[0018] The second modified primer is to molecularly modify the base sequence between the RBS (ribosome binding site) and the start codon of the pET vector. The modified vector fragment can be connected with the modified gene fragment to obtain a modified recombinant plasmid. This 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 of human lactoferrin in the prokaryotic expression system.

[0019] The gene sequence of the C lobe fragment of human lactoferrin is shown in SEQ ID NO: 2.

[0020] As an embodiment, in step (2), the first modified primers are dT-rhLF F' and rhLF R'; in step (3), the second modified primers are pET28 F' and pET28-dT;

[0021] The dT-rhLF F' sequence is shown in SEQ ID NO: 3, the rhLF R' sequence is shown in SEQ ID NO: 4, the pET28 F' sequence is shown in SEQ ID NO: 5, and the pET28-dT sequence is shown in SEQ ID NO: 6.

[0022] As another embodiment, in step (2), the first modified primers are dA-rhLF F' and rhLF R'; in step (3), the second modified primers are pET28 F' and pET28-dA R';

[0023] The dA-rhLF F' sequence is shown in SEQ ID NO: 7, the rhLF R' sequence is shown in SEQ ID NO: 4, the pET28 F' sequence is shown in SEQ ID NO: 5, and the pET28-dA R' sequence is shown in SEQ ID NO: 8.

[0024] As another embodiment, in step (2), the first modified primers are AT-rhLF F' and rhLF R'; in step (3), the second modified primers are pET28 F' and pET28-AT R';

[0025] The AT-rhLF F' sequence is shown in SEQ ID NO: 9, the rhLF R' sequence is shown in SEQ ID NO: 4, the pET28 F' sequence is shown in SEQ ID NO: 5, and the pET28-AT R' sequence is shown in SEQ ID NO: 10.

[0026] The amino acid sequence of recombinant human lactoferrin is shown in SEQ ID NO: 1.

[0027] The beneficial effects of the present invention are that: 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 method is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SDS-PAGE electrophoresis diagram of the expression of recombinant human lactoferrin in the strain induced by IPTG at different temperatures;

[0029] Figure 2 This is the SDS-PAGE electrophoresis diagram of recombinant human lactoferrin expressed by different transformed strains;

[0030] Figure 3 This is a comparison chart of the expression levels of recombinant human lactoferrin in different transformed strains;

[0031] Figure 4 It is the SDS-PAGE electrophoresis diagram of the samples collected at each stage of the nickel ion affinity column in the present invention;

[0032] Figure 5 It is the SDS-PAGE electrophoresis diagram of the samples collected at each stage of the Q ion column in the present invention;

[0033] Figure 6 Schematic diagram of the appearance of the recombinant human lactoferrin lyophilized powder (left) and the reconstituted solution (right) obtained by the present invention;

[0034] Figure 7 This is the SDS-PAGE electrophoresis diagram of the recombinant human lactoferrin obtained by the present invention;

[0035] Figure 8 This is a graph verifying the antioxidant activity of recombinant human lactoferrin;

[0036] Figure 9 is the plasmid map of pET28a;

[0037] Figure 10 This is the plasmid map of pET28g. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described in detail below through specific embodiments.

[0039] 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 examples, unless otherwise specified, are all conventional methods in the art.

[0040] Example 1: Expression of recombinant human lactoferrin

[0041] The C lobe fragment gene sequence of human lactoferrin (SEQ ID NO: 2) was inserted into pET28a (i.e. pET- 28a (+), plasmid map see Figure 9 ) plasmid, and the recombinant plasmid (pET28a-rhLF) identified by sequencing was transformed into Escherichia coli BL21. A single colony was picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C, 220 rpm in a shaking incubator. A 1.0% inoculation ratio of the overnight culture was added to LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C, 220 rpm in a shaking incubator. Cultures were grown until they entered the logarithmic growth phase, with an OD600 value of 0.4-0.6. IPTG was then added to a final concentration of 0.5 mM and induced overnight at 18°C, 25°C, and 37°C. The bacterial cultures were subjected to SDS-PAGE electrophoresis to determine the expression level of rhLF (recombinant human lactoferrin).

[0042] like Figure 1 As shown, compared with the uninduced strains, the strains induced by IPTG at three temperatures all showed obvious specific bands at 39.5 kDa, and the expression level was highest when induced at 37 °C. Therefore, the subsequent screening of high-expressing strains was carried out by induction at 37 °C.

[0043] Example 2: Vector optimization and screening of high-expressing strains

[0044] Using the pET28a-rhLF plasmid as a template, primers were designed (underlined bases are modified), see Table 1:

[0045] Table 1:

[0046] .

[0047] 2. Use primer pairs dT-rhLF F' / rhLF R'; dA-rhLF F' / rhLF R' and AT-rhLF F' / rhLF R' to perform PCR on plasmid pET28a-rhLF to obtain the target gene.

[0048] The PCR systems are shown in Tables 2-4:

[0049] Table 2 dT-rhLF F' / rhLF R' PCR system:

[0050] ;

[0051] Table 3 dA-rhLF F' / rhLF R' PCR system:

[0052] ;

[0053] Table 4 AT-rhLF F' / rhLF R' PCR system:

[0054] .

[0055] PCR procedure:

[0056] 1. Pre-denaturation: 98°C, 5 minutes;

[0057] 2. Loop 32 times:

[0058] Denaturation: 98°C, 15 seconds

[0059] Annealing: 58°C, 15 seconds

[0060] Extension: 72°C, 20 seconds;

[0061] 3. Final extension: 72°C, 5 minutes

[0062] 4. Keep warm: 4℃, unlimited time.

[0063] 10× Loading buffer (TaKaRa) was added to the PCR product and electrophoresis was performed on a 1% agarose gel at 150 V for 15 min. After that, the nucleic acid bands were observed and the target gene was recovered by cutting the gel.

[0064] 3. Use primer pairs pET28 F' / pET28-dT R', pET28 F' / pET28-dA R' and pET28 F' / pET28-AT R' to pET28g (plasmid map see Figure 10 ) were used to perform PCR to obtain the transformed vector.

[0065] In the present invention, the unmodified pET28a plasmid vector can be used, but the modified one cannot be used and another pET28g plasmid vector needs to be used. Analysis of the reason revealed that the pET28a plasmid has an NcoI nuclease cleavage site between the RBS and the start codon ATG ( Figure 9 The recognition sequence of the restriction enzyme site is CCATGG (SEQ ID NO: 11). If the NcoI nuclease site is retained, a single-base frameshift mutation will occur. The RBS of the pET28g plasmid is followed by the NdeI nuclease site ( Figure 10 As shown in the red box), the recognition sequence of this restriction site is CATATG (SEQ ID NO: 12), which is the first ATG after RBS. Using the pET28g plasmid will not cause frameshift mutations.

[0066] The PCR systems are shown in Tables 5-7:

[0067] Table 5 pET28 F' / pET28-dT R' PCR system:

[0068] ;

[0069] Table 6 pET28 F' / pET28-dAR' PCR system:

[0070] ;

[0071] Table 7 pET28 F' / pET28-AT R' PCR system:

[0072] .

[0073] PCR procedure:

[0074] 1. Pre-denaturation: 98°C, 5 minutes;

[0075] 2. Loop 32 times:

[0076] Denaturation: 98°C, 15 seconds

[0077] Annealing: 58°C, 15 seconds

[0078] Extension: 72°C, 1 min 40 s;

[0079] 3. Final extension: 72°C, 5 minutes;

[0080] 4. Keep warm: 4℃, unlimited time.

[0081] 10× Loading buffer (TaKaRa) was added to the PCR product and electrophoresis was performed on a 1% agarose gel at 150 V for 15 min. After that, the nucleic acid bands were observed and the gel was cut to recover the vector.

[0082] 4. Recombinantly ligate the recovered vector and target gene. Transform the recombinant ligation products into DH5α cells, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and incubate again on ice for 2-3 minutes. Add 500 μL of antibiotic-free LB medium and incubate at 37°C on a shaker for 40 minutes. Centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in 500 μL of antibiotic-free LB medium. Spread the resuspended bacteria on a Kana plate and incubate at 37°C overnight. The next day, pick 5-10 single colonies from a Kana plate (tryptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L, agar 15 g / L, kanamycin 50 μg / mL) and place each colony in 300 μL of LB medium containing 50 μg / mL kanamycin. Incubate on a shaker at 37°C, 220 rpm, for 3-4 hours. The resulting suspension is then used for PCR. According to the size of the target band, positive colonies were selected for sequencing, and the strains with complete sequencing results were selected to extract plasmids, which were dA, dT and AT respectively.

[0083] 5. Separately transform dA, dT, AT, and the unmodified plasmid (recombinant plasmid pET28a-rhLF prepared by the method in Example 1) into BL21 cells. Select the positive BL21 strain and inoculate it into 3 mL of LB liquid medium containing kanamycin (50 μg / mL). Incubate at 37°C for 4-5 h. Then, add IPTG to a final concentration of 0.5 mM and induce at 37°C overnight.

[0084] 6. Take the bacterial solution and perform SDS-PAGE electrophoresis. Use ImageJ software to analyze the expression level and select the strain with the highest expression level. Figure 2-Figure 3 As shown in the figure, compared with the strain before creation, dA, dT and AT modifications can effectively improve the expression level of rhLF in E. coli, among which AT modification has the most obvious improvement effect, so the AT strain was selected as the subsequent high-expression fermentation strain.

[0085] Example 3: Purification of recombinant human lactoferrin

[0086] 1. Add buffer A (50mM Na₂HPO₄, 300mM NaCl, 10% glycerol, 1mM DTT, pH 7.5) to the cells collected by centrifugation after expression of the AT strain. Resuspend the cells thoroughly by pipetting, then homogenize 3-4 times. Centrifuge at 11,000 rpm at 4°C for 30 minutes and retain the pellet. Add buffer B (20mM Tris, 8M urea, pH 8.0) to the pellet. Stir at room temperature for 1 hour and centrifuge at 11,000 rpm at 4°C for 30 minutes. Retain the supernatant and dilute it 10-fold with buffer C (20mM Tris, 100mM NaCl). Mix thoroughly and allow the supernatant to renature overnight at 4°C. Exchange the renaturation buffer and concentrate it into buffer D (20mM Tris, 100mM NaCl, pH 8.0) to obtain a sample solution containing recombinant human lactoferrin.

[0087] 2. Wash the nickel ion affinity column (Ni-NTA) with buffer D, then mix the column and sample solution and incubate, gently shake at room temperature or on ice for 30 minutes, and then load the column. Use a gradient of buffer D containing 150 mM and 500 mM imidazole to wash the impurities and elute the protein. Collect samples at each stage and run SDS-PAGE for detection. According to the gel image results ( Figure 4 ), collect the 150mM imidazole eluate (high target protein content), concentrate it and exchange it into buffer E (20mM Tris-HCl buffer, pH 8.0) for further purification. Wash the Q column (strong anion exchange chromatography column) with buffer E, then mix the column with the recombinant lactoferrin solution after exchange and concentration, and then load it onto the column. Wash the impurities and eluted protein with a gradient of buffer E containing 150mM, 200mM, 500mM, and 1M NaCl. Collect samples at each stage and perform SDS-PAGE gel testing. According to the gel map results ( Figure 5 ), collect the 150mM and 200mM NaCl eluates (the target protein has higher purity), exchange them for pure water, concentrate them and then lyophilize them.

[0088] like Figure 6 As shown, the recombinant human lactoferrin lyophilized powder obtained by the present invention has a reddish-brown appearance. When reconstituted 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 that ( Figure 7 ), the protein purity is about 88%, which is relatively high.

[0089] Example 4: Verification of the Antioxidant Activity of Recombinant Human Lactoferrin

[0090] The experimental group settings are shown in Table 8:

[0091] Table 8:

[0092] .

[0093] The details are as follows:

[0094] 1. Culture BALB / 3T3 cells in a 37°C, 5% CO2 cell culture incubator. Subculture cells when they grow to 80-90% of the flask. Use complete culture medium [10% fetal bovine serum + 90% DMEM medium (Gibco)] to culture cells at a rate of 5×10 3 Each group of cells was seeded with three replicates in a 96-well plate and cultured at 37°C with 5% carbon dioxide for 24 hours.

[0095] 2. Discard the supernatant from each well and add 100 μL of maintenance medium [0.4% fetal bovine serum + 99.6% DMEM (Gibco)] to each well, along with 100 μL of native lactoferrin and recombinant human lactoferrin obtained in Example 3, to a final concentration of 0.3 mg / mL. Incubate at 37°C and 5% CO2 for 24 hours. For the blank control and hydrogen peroxide groups, only maintenance medium was added.

[0096] 3. Discard the supernatant from each well (including the maintenance medium and test sample) and add 100 μL of hydrogen peroxide prepared in the maintenance medium to each well to a final concentration of 150 μM. Incubate at 37°C, 5% CO2 for 2 hours. For the blank control group, only the maintenance medium should be added.

[0097] 4. Discard the supernatant (including maintenance medium and hydrogen peroxide) from the wells and add 100 μL of a fluorescent probe for reactive oxygen species detection (Biyuntian, Catalog No. S0033S) prepared in maintenance medium to each well. Incubate at 37°C, 5% CO2 for 20 minutes. Carefully aspirate the supernatant (including maintenance medium and reactive oxygen species detection probe) from the wells and wash 2-3 times with maintenance medium. Finally, add 100 μL of maintenance medium to each well. Place the wells in a microplate reader and measure fluorescence intensity using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0098] like Figure 8 As shown, compared with natural lactoferrin, the recombinant human lactoferrin of the present invention can also reduce the level of reactive oxygen species caused by hydrogen peroxide, and the effect is even better, indicating that it has strong antioxidant activity.

[0099] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

[0100] SEQ ID NO: 1:

[0101] MHHHHHHDDDDKVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK;

[0102] SEQ ID NO:2:

[0103]

Claims

1. A method for preparing recombinant human lactoferrin based on an Escherichia coli vector, characterized in that: The following steps are involved: (1) Recombinant expression of the recombinant human lactoferrin shown in SEQ ID NO: 1 was performed, and the gene sequence of the recombinant 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 plasmid pET28g as a template, design the second modified primer for PCR to obtain the modified vector; (4) Recombinantly connecting the modified target gene obtained in step (2) with the modified vector obtained in step (3), transferring the recombinant connection product into DH5α, culturing, extracting, and obtaining a recombinant plasmid; (5) The recombinant plasmid was transformed into Escherichia coli BL21 to obtain an Escherichia coli engineered strain that efficiently expressed recombinant human lactoferrin; (6) The engineered Escherichia coli strain that efficiently expresses recombinant human lactoferrin is fermented and cultured to induce the expression of recombinant human lactoferrin; the expression product is purified to obtain recombinant human lactoferrin; the amino acid sequence of the recombinant human lactoferrin is shown in SEQ ID NO:

1.

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

6.

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

8.

5. The preparation method according to claim 1, characterized in that In step (2), the first modified primers are AT-rhLFF' and rhLF R'; in step (3), the second modified primers are pET28 F' and pET28-AT R'; The AT-rhLF F' sequence is shown in SEQ ID NO: 9, the rhLF R' sequence is shown in SEQ ID NO: 4, the pET28 F' sequence is shown in SEQ ID NO: 5, and the pET28-AT R' sequence is shown in SEQ ID NO: 10.

Images