Recombinant engineering bacteria expressing fetuin B and application thereof

Abstract

This invention discloses a recombinant engineered bacterium expressing fetuin B (FetuB) and its applications, relating to the field of genetic engineering technology. The recombinant engineered bacterium of this invention uses Pichia pastoris as the host bacterium to introduce fetuin B (FetuB). FetuB Following gene derivation, efficient expression of fetoglobulin B in Pichia pastoris was achieved by optimizing codons and improving fermentation conditions. Under shake-flask culture conditions, the expression levels were 320±25 mg / L for CBS7435, 280±30 mg / L for SMD116, and 580±45 mg / L for SuperMan5. After optimizing fermentation conditions, the highest yield in fermenter culture reached 27.35 g / L. The fetoglobulin B of this invention exhibits good biological activity, high expression, and a simple operation process, making it suitable for large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium expressing fetuin B (FetuB) and application thereof. BACKGROUND

[0002] Fetuin-B is a kind of glycoprotein mainly secreted by liver, and its research in the fields of metabolic diseases, cardiovascular diseases, inflammation and reproductive system diseases has gradually increased in recent years. Current studies have shown that fetuin-B has the following important biological effects: (1) promoting cell endocytosis: fetuin-B enhances the endocytosis capacity of cells to specific substances by regulating cell membrane fluidity and signal transduction, and plays a role in nutrient uptake and metabolic waste removal; (2) regulating cell proliferation and differentiation: as a multifunctional glycoprotein, fetuin-B participates in the synergistic effect of cell growth factors, affects the cell proliferation cycle and differentiation process, and is particularly significant in embryonic development and tissue repair; (3) maintaining calcium and phosphorus metabolism balance: fetuin-B can bind with free calcium ions to inhibit abnormal calcium salt deposition and prevent soft tissue calcification, which is crucial for bone metabolism homeostasis; (4) inflammation and metabolism regulation: in chronic liver disease models, fetuin-B reduces inflammation and fibrosis progression by inhibiting the activity of pro-inflammatory factors (such as TNF superfamily members); at the same time, it participates in the regulation of insulin sensitivity and affects glucose and lipid metabolism. Fetuin-B has been widely used in the prediction of cardiovascular disease risk, the regulation of metabolic syndrome and the regulation of chronic liver disease progression.

[0003] Currently, the fetuin sources on the market mainly include bovine fetuin B extracted from bovine placental serum and recombinant human fetuin B expressed by mammalian cells, and the research on recombinant fetuin B expressed in microorganisms is still relatively rare. SUMMARY

[0004] Based on the problems existing in the prior art and filling the gap in the prior art, the present application provides a recombinant engineering bacterium expressing fetuin B (FetuB) and application thereof, and the content of the present application is as follows:

[0005] In a first aspect, the present application provides a recombinant engineering bacterium expressing fetuin B (FetuB), which contains an expression vector connected with a target gene, wherein the target gene is a fetuin B (FetuB) gene, and the amino acid sequence of the fetuin B (FetuB) gene is shown as SEQ ID NO. 2. FetuB ) gene, preferably,

[0006] Further, the fetuin B (FetuB) gene is a gene encoding the amino acid sequence shown as SEQ ID NO. 2, preferably, FetuB ) gene, preferably, FetuBThe nucleotide sequence of the gene is shown as SEQ ID NO. 1 or has 90% homology with the nucleotide sequence shown as SEQ ID NO. 1.

[0007] Further, the recombinant engineering bacteria is selected from one or more of Pichia pastoris, Hansenula, Candida, Saccharomyces cerevisiae. Preferably, the recombinant engineering bacteria is Pichia pastoris. Further, the recombinant engineering bacteria is at least one of Pichia pastoris CBS7435, SMD116, SuperMan5.

[0008] Further, the expression vector of the recombinant engineering bacteria includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZ alpha, pGAP alpha.

[0009] Further, the promoter of the recombinant engineering bacteria includes any one or more of AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter and AOX2 promoter.

[0010] In a specific embodiment of the present application, the expression vector is pPICZ alpha and the promoter is AOX1 promoter.

[0011] In a second aspect of the present application, the recombinant engineering bacteria is provided for use in the preparation of Fetu B and / or for increasing the yield of Fetu B.

[0012] In a third aspect of the present application, a method for preparing Fetu B is provided, which comprises the step of culturing and fermenting the recombinant engineering bacteria to obtain Fetu B.

[0013] Further, the method specifically comprises the following steps: inoculating the recombinant engineering bacteria into BMGY culture medium and culturing to OD 600 =5~7, resuspending the cells in 40 mL BMY culture medium, adding methanol to the shake flask every 24 h to maintain induction, and collecting the culture after 72 h of induction.

[0014] Further, the concentration of the methanol is 0.5%~1.2% (v / v) in the final concentration in the culture solution; and the culture temperature is 24~30℃.

[0015] The beneficial effects of the present application include but are not limited to:

[0016] The present application improves the yield of fetuin B by increasing the copy number of exogenous genes, introducing alpha mating factor signal peptide, optimizing the nucleotide sequence of the target gene, and optimizing the culture conditions of Pichia pastoris, so that fetuin B is efficiently expressed in Pichia pastoris strains. The expression amounts of different strains in 72 h shake flasks are compared, and the expression amount of the glycosylation optimized SuperMan5 can reach 580±30 mg / L; the expression amount of the protease deletion type SMD116 is 280±30 mg / L, and the expression amount of the wild type CBS7435 is 320±25 mg / L. After optimizing the fermentation conditions, the yield in the fermenter can reach 27.35 g / L. The recombinant engineering bacteria show strong potential in improving the yield of high economic value proteins, and have broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0017] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0018] Figure 1 is a Fetuin-B map of pPICZ in the embodiment of the present application;

[0019] Figure 2 is a 72h protein induction OD map in the embodiment of the present application 600 Comparison chart;

[0020] Figure 3 is a comparison chart of electrophoresis before and after plasmid digestion in the embodiment of the present application, wherein 1: Marker: 250-500-750-1000-1500-2000-2500-3000-4000-5000-6000-8000-10000, 2: uncut FetuB-pPICZα plasmid, 3: BglII single-digested FetuB-pPICZα plasmid (250 ng / μl);

[0021] Figure 4 is a comparison chart of shake flask induction fermentation SDS-PAGE in the embodiment of the present application, from left to right, CSB7435, SMD116, SuperMan5, Marker;

[0022] Figure 5 is a comparison chart of fermentation condition optimization in the embodiment of the present application. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. In addition, the raw materials used in the embodiments of the present application are commercially available unless otherwise specified.

[0024] The present application will be further described in conjunction with specific embodiments, and the innovative points and features of the present application will become more apparent with the description. However, these embodiments are only illustrative, and do not constitute any limitation on the scope of the present application. It should be understood by those skilled in the art that modifications or substitutions can be made to the details and forms of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of protection of the present application.

[0025] LB solid medium: 10 g / L Tryptone, 5 g / L Yeast Extract, 10 g / L NaCl, sterilized at 121℃ for 20 min, used for the culture of E. coli;

[0026] YPD liquid medium: 20 g / L Tryptone, 10 g / L Yeast Extract, 2% glucose, sterilized at 115℃ for 25 min, used for the culture of P. pastoris;

[0027] YPD solid medium: 20 g / L agar powder and 1‰ zeocin were added to the YPD liquid medium, used for the monoclonal growth of P. pastoris;

[0028] BMY (1 L): 1% yeast extract, 2% Tryptone, 100 mM potassium phosphate pH 6.0, 10% YNB, 0.2% biotin, 1‰ methanol;

[0029] BMGY (1 L): 1% yeast extract, 2% Tryptone, 100 mM potassium phosphate pH 6.0, 10% YNB, 0.2% biotin, 1‰ methanol, 2% glycerol.

[0030] Example 1 Construction of pPICZα-Fetuin-B expression plasmid

[0031] Synthesis of fetuin B gene: The amino acid sequence of human fetuin B (SEQ ID NO: 2) was searched in Uniprot, and the DNA coding sequence of fetuin B was optimized by codon optimization software to be the codon preference of P. pastoris to obtain the DNA sequence encoding the fetuin B of the present application SEQ ID NO. 1 (wherein the nucleotide sequence represented by SEQ ID NO. 1 contains enzyme cutting sites at both ends, and the sequences are GAATTC and GCGGCCGC ), and the full sequence was synthesized by Shanghai Generay Biotech Co., Ltd. and integrated into the multiple cloning site of pPICZα vector.

[0032] Construction and identification of recombinant plasmid: as Figure 1The synthesized fetuin B sequence was inserted between EcoR I and Sal I sites, expressed under the control of AOX1 promoter, and fused with an α-factor that can be cleaved at the N terminus.

[0033] The DNA coding sequence of fetuin B (GenBank: 26998) was optimized (SEQ ID NO. 1), digested with EcoRI and SalI, separated by 1% agarose gel electrophoresis, purified and recovered, and linked with T4 DNA ligase overnight. The synthesized gene was cloned into the yeast expression vector pPICZα to obtain the pPICZα-Fetuin-B plasmid. The next day, the ligation product was used to transform E. coli DH5α competent cells, which were plated on LB plates containing zeocin and incubated at 37°C overnight. The next day, single colony liquid was picked from the plate, and the recombinant plasmid was extracted after amplification and identified by double digestion with EcoR I and Snal I. A band with a size consistent with the length of the target gene sequence (4500 bp) was obtained (as shown in Figure 3 ), indicating that the expression plasmid was correctly constructed.

[0034] Example 2: Screening of positive transformants

[0035] A rapid plasmid extraction kit CW2619M (purchased from Kangwei Century)

[0036] According to the instructions of the rapid plasmid extraction kit CW2619M, a single colony was inoculated in 5 mL of LB culture containing zeocin and incubated at 37°C for 16-20 h. Then, 600 μL of the overnight culture was transferred to an EP tube, 100 μL of Buffer L2 was added, and the tube was gently inverted 8 times. Then, 350 mL of Buffer N3 (RNase A) was added, and the tube was inverted 10 times. After centrifugation at 13000 rpm for 3 min, 1 mL of supernatant was transferred to a new EP tube. Then, 500 μL was transferred to an adsorption column, which was centrifuged at 13000 rpm for 15 s, and the solution was discarded. Then, 150 μL of Buffer PB was added, and the column was centrifuged at 13000 rpm for 15 s, and the solution was discarded. Then, 400 μL of Buffer PW (EtOH) was added, and the column was centrifuged at 13000 rpm for 1 min, and the solution was discarded. After air-drying for 10 min, 80 μL of 70°C dd H2O was added, and the column was centrifuged at 13000 rpm for 1 min. The Nanodrop was measured, and the plasmid was used for linearization experiments.

[0037] Linearization of recombinant plasmid

[0038] Table 1: Linearization system of recombinant plasmid

[0039]

[0040] The linearization system of recombinant plasmid is shown in Table 1, and the reaction conditions are as follows: 37 ℃ water bath for 10 min; 65 ℃ water bath for 20 min.

[0041] The DNA product purification kit CW2301M (purchased from Kangwei Century) was used to purify the linearized plasmid

[0042] Column equilibration: 500 μL BL was added to CB2, centrifuged at 12000 rpm for 1 min, and the solution was discarded. 5 times the volume of PB was added to the enzyme digestion system, mixed well. The solution was transferred to CB2, RT for 2 min, centrifuged at 12000 rpm for 1 min, the solution was discarded, and the operation was repeated once. Centrifuged at 12000 rpm for 2 min again, the solution was discarded; air-dried for 10 min, 40 μL 70 ℃ dd H2O was added, RT for 2 min, centrifuged at 12000 rpm for 2 min, and Nanodrop was measured for electroporation experiment.

[0043] Pichia pastoris strains CBS7435, SMD116, SuperMan5 were electroporated

[0044] 5-10 μg of linearized recombinant plasmid was added to 80 μL of CBS7435 / SMD116 / SuperMan5 competent cells, mixed well, added to the electroporation cup, and placed in ice bath for 5 min; 1500 V-25 μF-200 Ω, electroporation; the bacterial solution was transferred to 1 mL of ice sorbitol solution, mixed well, incubated at 30 ℃, 225 rpm for 1 h, 50-100 μL was taken and plated on YPD plates containing zeocin resistance, and cultured at 30 ℃ for 48-72 h.

[0045] Screening of positive transformants (the kit used was yeast genomic DNA extraction kit DP307-02 purchased from Tiangeng)

[0046] According to the instructions of the yeast genomic DNA extraction kit, the experiment was carried out, specifically: take the single colony on the zeocin-resistant plate and add it to 5 mL YPD medium, 220 rpm, 30°C overnight culture; take 1 mL of bacterial liquid after culture, centrifuge at 12000 rpm for 1 min, discard the supernatant; add 600 μL sorbitol, 5 μL 50 U Lyticase, mix well, 30°C water bath for 30 min, centrifuge at 4100 rpm for 10 min, discard the supernatant; add 200 μL GA and mix well, add 4 μL RNase A, shake for 15 s, RT for 5 min; add 20 μL Proteinase K, mix well; add 220 μL GB, mix well, 70°C water bath for 10 min, centrifuge at 3000 rpm for 3 s; add 220 μL EtOH, mix well, centrifuge at 3000 rpm for 3 s; transfer the solution in the EP tube to CB3, centrifuge at 12000 rpm for 30 s, discard the solution; add 500 μL GD (EtOH), centrifuge at 12000 rpm for 30 s, discard the solution; add 600 μL PW (EtOH), centrifuge at 12000 rpm for 30 s, discard the solution, repeat the operation once; centrifuge at 12000 rpm for 2 min again, discard the solution; air dry for 10 min, add 50 μL 70°C dd H2O, RT for 2 min, centrifuge at 120000 rpm for 2 min. Then perform PCR and gel electrophoresis verification.

[0047] Example 3 Shake flask induction screening of strains with high expression

[0048] In order to screen the single clone of high expression of Fetuin-B, the positive transformants of different strains were transferred to 40 mL BMGY solution, 30°C, 220 rpm overnight culture, the OD value was adjusted to 6, the cells were resuspended in 40 mL BMY medium, 160 μL pure methanol was added, the final concentration of methanol in the culture solution was 1% (v / v), 25°C, 220 rpm, 160 μL pure methanol was added every 24 h of culture, and the methanol concentration was maintained at 1% (v / v), the OD value was measured every 24 h, and the results are shown in Table 2. 600 600 , the results are shown in Table 2, until the end of 72 h, take 1 mL of bacterial liquid, centrifuge at 10000 rpm for 5 min, collect the supernatant and detect SDS-PAGE (as shown in Figure 4 ), the results are shown in Table 3, according to Figure 2 , the 72 h induction results show that the OD value of SuperMan5 d is 600 ​The highest value, combined with Table 3 shows that the SuperMan5 protein expression is the highest, 72 h can reach 580 ± 45 mg / L, select the highest expression of the positive strain of SuperMan5 transformant for subsequent production.

[0049] Table 2 Protein induction OD 600 Comparison table

[0050]

[0051] Table 3 Protein expression data table

[0052]

[0053] Example 5 DNA sequence optimization

[0054] According to the amino acid sequence of FetuB, five groups of DNA sequences are designed for comparison, and the sequence with the highest expression (sequence as SEQ ID NO. 3-7) is selected. The results are shown in Table 4. The DNA sequence of the strain with the highest expression is SEQ ID NO. 1, which is used for subsequent fermenter culture.

[0055] Table 4 Different DNA sequence culture expression

[0056]

[0057] Example 4 Fermenter culture

[0058] 1. Preparation of seed liquid:

[0059] Take the preserved strain SuperMan5- FetuB Plate streaking, incubate at 30°C for 2-3 d, pick well-grown single colonies and inoculate in 10 mL YPD liquid medium, incubate at 30°C, 220 rpm for about 24 h. Take 5 mL of the above culture for further expansion, inoculate in 500 mL YPD liquid medium, incubate at 30°C, 220 rpm, when the shake flask seed OD 600 Reaches 10, as the seed liquid for feeding.

[0060] 2. Batch fermentation:

[0061] Put 20 L BSM medium into the fermentor, adjust pH to 6.0 with ammonia water, sterilize at 121℃ for 30 min, correct the dissolved oxygen to 0% after cooling, set the temperature to 30℃, the aeration rate to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 6.0 again. Discard about 500 mL of the medium in the fermentor through the sampling port to make the volume in the tank about 19.5 L before inoculation. Pump the seed liquid in the shake flask into the fermentor and correct the dissolved oxygen to 100%, set the rotation speed to 300 rpm. The dissolved oxygen control mode is selected as "rotation speed linkage", and the dissolved oxygen is maintained above 20% by adjusting the rotation speed (300 rpm-1000 rpm) and the aeration rate during fermentation.

[0062] 3. Glycerol fed-batch fermentation:

[0063] When the dissolved oxygen suddenly increases and the rotation speed suddenly decreases, enter the glycerol fed-batch fermentation stage, and add glycerol at a flow rate of 15 mL / h·L, and the tank parameters remain unchanged.

[0064] 4. Methanol fed-batch fermentation:

[0065] Stop adding glycerol at 30 h (wet weight about 207 g / L), and the dissolved oxygen quickly rises, entering the methanol fed-batch fermentation stage (methanol concentration maintained at 0.5%-1.2% (v / v)). No carbon source is added in the tank for 1 h to ensure complete consumption of glycerol. After the starvation treatment, add methanol at a constant flow rate of 10 mL / h·L, and the tank parameters remain unchanged. Real-time monitoring of each fermentation parameter is required to maintain the dissolved oxygen above 20%. The fermentation is stopped at 132 h, and the fermentor is discharged. The fermentation product is centrifuged at 8000 rpm for 20 min, and the supernatant is collected and stored at -20℃.

[0066] Example 5. Optimization of culture conditions

[0067] 1. Preparation of seed liquid:

[0068] Take the preserved strain SuperMan5- FetuB Perform plate streaking and incubate at 30℃ for 2-3 d, pick well-grown single colonies and inoculate in 10 mL of YPD liquid medium, and incubate at 24℃ / 30℃ at 220 rpm for about 24 h. Take 5 mL of the above culture for further expansion, inoculate in 500 mL of YPD liquid medium, and incubate at 24℃ / 30℃ at 220 rpm. When the OD 600 of the seed liquid reaches 10, it is used for inoculation.

[0069] 2. Batch fermentation:

[0070] Put 20 L BSM medium into the fermentor, adjust pH to 5.0 / 6.0 with ammonia water, sterilize at 121 ℃ for 30 min, correct the dissolved oxygen to 0% after cooling, set the temperature to 30 ℃, the aeration rate to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87 mL of sterilized PTM1 solution to the medium, and adjust the pH of the medium to the same pH value as the ammonia water. Discard about 500 mL of the medium in the fermentor through the sampling port to make the volume in the tank about 19.5 L before inoculation. Pump the seed culture in the shake flask into the fermentor, and correct the dissolved oxygen to 100%, set the rotation speed to 300 rpm. Select "rotation speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 20% by adjusting the rotation speed (300 rpm-1000 rpm) and the aeration rate during fermentation.

[0071] 3. Glycerol fed-batch fermentation:

[0072] When the dissolved oxygen suddenly increases and the rotation speed suddenly decreases, enter the glycerol fed-batch fermentation stage, and add glycerol at a flow rate of 15 mL / h·L, and the tank parameters remain unchanged.

[0073] 4. Methanol fed-batch fermentation:

[0074] Stop adding glycerol when the fermentation reaches 30 h (wet weight reaches about 207 g / L), the dissolved oxygen quickly rises, and enter the methanol fed-batch fermentation stage (the methanol concentration is maintained at 0.5%-1.2% (v / v)). No carbon source is added in the tank for 1 h to ensure that the glycerol is completely consumed. After the starvation treatment, add methanol at a constant flow rate of 10 mL / h·L, the tank parameters remain unchanged, and the fermentation parameters are monitored in real time to maintain the dissolved oxygen above 20%. The fermentation is stopped at 132 h, and the fermentor is discharged. The fermentation product is centrifuged at 8000 rpm for 20 min, the supernatant is collected, and stored at -20 ℃.

[0075] 5. The strain 1 condition is a culture temperature of 24 ℃, and the solution pH is adjusted to 5.0 with ammonia water;

[0076] The strain 2 condition is a culture temperature of 24 ℃, and the solution pH is adjusted to 6.0 with ammonia water;

[0077] The strain 3 condition is a culture temperature of 30 ℃, and the solution pH is adjusted to 5.0 with ammonia water;

[0078] The strain 4 condition is a culture temperature of 30 ℃, and the solution pH is adjusted to 6.0 with ammonia water.

[0079] 6. As shown in Figure 5 The fermentation results show that the fermentation condition of SuperMan5-FetuB is a culture temperature of 24 ℃, the solution pH is adjusted to 6.0 with ammonia water, and the protein expression value is the highest.

[0080] The above merely describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but determined by the claims of the present application. The present application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical thought and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant engineered bacteria expressing Fetuin B (FetuB) characterized in that, The recombinant engineering bacteria contain an expression vector connected with a target gene, the target gene is fetoprotein B FetuB ) gene, the amino acid sequence of which is shown as SEQ ID NO. 2, the nucleotide sequence of the fetoprotein B FetuB ) gene is shown as SEQ ID NO. 1, and the recombinant engineering bacteria are Pichia pastoris SuperMan5.

2. The recombineering bacteria of claim 1, wherein, The expression vector of the recombinant engineering bacteria comprises any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, pGAPα.

3. The recombineering bacteria of claim 1, wherein, The promoter of the recombinant engineering bacteria comprises any one or more of AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter and AOX2 promoter.

4. Use of the recombinant engineering bacteria according to any one of claims 1-3 in the preparation of fetuin B (FetuB) and / or in the improvement of fetuin B (FetuB) production.

5. A method of preparing Fetu B, characterized in that, The method comprises the step of culturing the recombinant engineering bacteria according to any one of claims 1-3 to obtain fetuin B (FetuB).

6. The method of claim 5, wherein, The method specifically comprises the following steps: inoculating the recombination engineering bacteria according to any one of claims 1-3 into a BMGY culture medium to culture to OD 600 =5~7, resuspending the cells in the BMY culture medium, adding methanol into the shake flask every 24 h to maintain induction, wherein the final concentration of the methanol in the culture solution is 0.5%~1.2% (v / v); the culture temperature is 30°C, and the pH is 6.0.

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