CN117924435A - Pichia pastoris for secretory expression of immunomodulatory peptide Y6, and construction method and application thereof - Google Patents
Pichia pastoris for secretory expression of immunomodulatory peptide Y6, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses pichia pastoris for secretory expression of immunoregulatory peptide Y6, and a construction method and application thereof. According to the invention, codon optimization is carried out on the gene of the immunoregulatory peptide Y6 according to the genetic codon preference of pichia pastoris, the optimized gene is directionally cloned into a pichia pastoris eukaryotic expression vector pPIC9K to construct recombinant expression plasmid, the recombinant expression plasmid is transformed into pichia pastoris GS115 competent cells, high-efficiency expression strains are screened, high-density fermentation culture is carried out on the engineering strains for efficiently expressing the immunoregulatory peptide Y6 in a 5L fermentation tank, and fermentation parameters and processes of the heterologously expressed immunoregulatory peptide are optimized. The invention takes pichia pastoris as an expression host for the first time, realizes the practical heterologous expression technology of the immunoregulatory peptide Y6, has good stability, high expression quantity and broad-spectrum antibacterial activity, and the construction method has simple operation and low cost, and the optimized fermentation process is suitable for large-scale production.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to pichia pastoris for secretory expression of immune regulating peptide Y6, and a construction method and application thereof.
Background
The immunoregulatory peptide is used as an important component of the immune system of the organism, and can directly kill pathogens or regulate the immune function of the organism. Unlike antibiotics, which interfere with the metabolic processes of bacteria, immunomodulatory peptides bind to bacterial membranes mainly by electrostatic action and physically destroy the bacterial membrane structure, resulting in bacterial death, and resistance to drugs is not easily generated. Functional studies show that the immunoregulatory peptide has broad-spectrum bactericidal activity, has no toxicity to mammalian cells and has potential therapeutic value. In addition, the immunoregulation peptide can improve the immunity and growth performance of animals, and is hopeful to be developed into a novel safe and efficient feed additive. However, the current immunoregulatory peptide adopts a chemical synthesis method, has complex steps, low yield and high cost, and limits the wide application of the immunoregulatory peptide in actual production.
The pichia pastoris expression system is one of the commonly used eukaryotic expression systems, and has the following advantages: 1) Has modifications in protein processing, folding, and post-translational; 2) The heterologous expression efficiency is high, the heterologous protein is not secreted basically, and the separation and purification of the target protein are facilitated; 3) The high-density culture can be realized in a simple culture medium, and the glycerol and the methanol can be used as the only carbon sources to produce the exogenous protein; 4) The exogenous gene can be stably integrated at a specific site in its genome; 5) The fermentation and purification operation is simple and convenient, and the industrial production is easy.
Disclosure of Invention
The invention aims to provide pichia pastoris for secretory expression of immune regulation peptide Y6, and a construction method and application thereof.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a gene encoding an immunomodulatory peptide Y6, the nucleotide sequence of which is shown in SEQ ID NO. 1.
In the invention, the amino acid sequence of the immunoregulatory peptide Y6 is shown as SEQ ID NO. 2.
In a second aspect, the invention provides biological materials comprising the genes, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria.
In a third aspect, the present invention provides a recombinant plasmid comprising the gene, wherein the starting vector of the recombinant plasmid is pPIC9K.
In a fourth aspect, the invention provides a method for constructing pichia pastoris for secretion expression of immunomodulatory peptide Y6, comprising: and (3) carrying out codon optimization on the encoding gene of the immunoregulatory peptide Y6 according to the preference of the genetic code of pichia pastoris, cloning the optimized gene into a pichia pastoris expression vector to construct a recombinant expression plasmid, converting the recombinant expression plasmid into pichia pastoris competent cells, and screening high-efficiency expression strains.
Further, the expression vector is pPIC9K, and the pichia competent cell is GS115.
Further, the nucleotide sequence of the optimized gene is shown as SEQ ID NO. 1.
In a fifth aspect, the invention provides a pichia pastoris engineered strain constructed according to the above method.
In a sixth aspect, the invention provides the use of an immunomodulatory peptide Y6 or an engineered bacterium as described in the manufacture of a broad spectrum antibacterial medicament for the treatment of infections caused by bacteria.
Such bacteria include, but are not limited to, E.coli (ESCHERICHIA COLI) ATCC25922, K88, K99, salmonella typhimurium (Salmonella Typhimurium) SL1344, ATCC14028, citrobacter typhimurium (Citrobacter rodentium) DBS100, ATCC51459, staphylococcus aureus (Staphylococcus aureus) ATCC6538, ATCC25923, CVCC1882.
In a seventh aspect, the present invention provides the use of an immunomodulatory peptide Y6 or an engineered bacterium thereof for the manufacture of a feed additive for preventing or treating diarrhea in an animal, for improving growth performance or immune function in an animal; the feed additive has the function of resisting gastrointestinal digestion degradation.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
According to the invention, codon optimization is carried out on the gene of the immunoregulatory peptide Y6 according to the genetic codon preference of pichia pastoris, the optimized gene is directionally cloned into a pichia pastoris eukaryotic expression vector pPIC9K to construct recombinant expression plasmid, the recombinant expression plasmid is transformed into pichia pastoris GS115 competent cells, high-efficiency expression strains are screened, high-density fermentation culture is carried out on the engineering strains for efficiently expressing the immunoregulatory peptide Y6 in a 5L fermentation tank, and fermentation parameters and processes of the heterologously expressed immunoregulatory peptide are optimized. The invention takes pichia pastoris as an expression host for the first time, realizes the practical heterologous expression technology of the immunoregulatory peptide Y6, has good stability, high expression quantity and broad-spectrum antibacterial activity, and the construction method has simple operation and low cost, and the optimized fermentation process is suitable for large-scale production.
Drawings
FIG. 1 is a schematic diagram showing the structure of a recombinant plasmid pPIC9K-Y6 according to a preferred embodiment of the present invention.
FIG. 2 is a diagram showing PCR verification of recombinant plasmids in a preferred embodiment of the present invention.
FIG. 3 is a diagram showing PCR verification of recombinant strains according to a preferred embodiment of the present invention.
FIG. 4 shows the OD 600, wet cell weight and total protein concentration of the high-density fermentation curve according to the preferred embodiment of the present invention.
FIG. 5 is a diagram showing the zone of inhibition of the high-density fermentation supernatant in a preferred embodiment of the present invention.
FIG. 6 is a diagram showing SDS-PAGE of purified immunomodulatory peptide Tris-Tricine according to the preferred embodiment of the present invention.
Detailed Description
The invention aims to provide pichia pastoris for secretory expression of immune regulation peptide Y6, and a construction method and application thereof.
The invention adopts the following technical scheme:
S1: optimizing and synthesizing an immunomodulatory peptide Y6 gene sequence;
S2: constructing and identifying a recombinant immunomodulatory peptide Y6 expression vector;
S3: construction of Pichia pastoris for secretion expression of the immunomodulatory peptide Y6;
S4: identifying the phenotype of the recombinant pichia pastoris transformant;
S5: screening recombinant pichia pastoris for efficiently expressing the immunoregulatory peptide Y6;
s6: high-density fermentation of recombinant pichia pastoris for efficiently expressing immunomodulatory peptide Y6;
s7: detecting antibacterial activity of the fermentation supernatant;
S8: purifying and preparing recombinant immunomodulatory peptide Y6;
S9: stability test of purified recombinant immunomodulatory peptide Y6.
The invention also provides application of the Pichia pastoris for secreting and expressing the immunoregulatory peptide Y6, which is used for inhibiting common pathogenic microorganisms.
The invention also provides application of the pichia pastoris for up-secretion expression of the immunoregulatory peptide Y6 in preparing broad-spectrum antibacterial medicines for treating infection caused by bacteria, feed additives for preventing or treating animal diarrhea and feed additives for improving animal growth performance or immune function.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 optimization and Synthesis of the sequence of the immunomodulatory peptide Y6 Gene
6 Histidines (His) are introduced at the tail end of the amino acid sequence of the immunoregulatory peptide, and the coding gene is optimized according to the preference of pichia pastoris codons. Introducing a restriction enzyme site EcoRI at the 5 'end of the sequence, introducing a restriction enzyme site NotI at the 3' end of the sequence, and artificially synthesizing the optimized immunoregulatory peptide gene by the Biotechnology Co-Ltd in Suzhou Jin Weizhi, wherein the nucleotide sequence of the optimized immunoregulatory peptide gene is shown as SEQ ID NO. 1.
EXAMPLE 2 construction and identification of recombinant immunomodulatory peptide Y6 expression vector
The artificially synthesized immunomodulatory peptide gene and pPIC9K in example 1 were digested by restriction enzymes EcoRI and NotI, respectively, and then recovered by 1.5% agarose gel electrophoresis, and the recovered fragments were linked by T4 ligase and transformed into E.coli DH 5. Alpha. Competent cells, and plated overnight for culture. The next day single colonies were picked for colony PCR verification, agarose gel electrophoresis results were as shown in FIG. 2, and sequencing verification was performed on recombinant plasmid pPIC9K-Y6 (structure schematic diagram see FIG. 1).
The specific colony PCR reaction system and procedure were as follows:
① The reaction system:
② Amplification procedure:
③ Primer sequences (5 '-3'):
5AOX：CTGGTTCCAATTGACAAGC
3AOX：TGGCATTCTGACATCCTC
EXAMPLE 3 construction of Pichia pastoris for secretion expression of immunomodulatory peptide Y6
Linearizing the recombinant plasmid by using restriction enzyme Sac I, adding the recombinant plasmid into pichia pastoris GS115 competent cells, lightly mixing the recombinant plasmid, transferring the recombinant plasmid to a precooled electrotransfer cup, carrying out ice bath for 5 minutes, transferring the recombinant plasmid to an electrotransfer instrument, and setting electrotransfer parameters: the voltage was 1.5kV, the resistance was 250Ω, the capacitance was 25μF, 1mL of 1M precooled sorbitol was added immediately after the electrotransformation, the mixture was allowed to stand in an incubator at 30℃for 1 hour, the mixture was spread on YPDS solid medium containing the corresponding antibiotics, and the mixture was incubated at 37℃for 3 days, and single colonies were picked for PCR identification, and the results were shown in FIG. 3.
Specific colony PCR reaction systems and procedures are shown below:
① The reaction system:
② Amplification procedure:
③ Primer sequences (5 '-3'):
5AOX：CTGGTTCCAATTGACAAGC
3AOX：TGGCATTCTGACATCCTC
EXAMPLE 4 identification of the phenotype of recombinant Pichia transformants
Positive transformants obtained by transformation in example 3 were inoculated onto MD and MM plates, respectively, and their methanol utilization phenotype (Mut+/Muts) was identified. Transformants capable of normal growth in both plates were His+/mut+ type; the growth was normal on MD plates, whereas on MM plates the growth was either not or slow, his+/Muts type.
EXAMPLE 5 screening of recombinant Pichia pastoris for efficient expression of immunomodulatory peptide Y6
The His+/mut+ transformants identified in example 4 were inoculated with a single colony in 10ml YPD medium and cultured at 30℃at 250rpm for 24h; the bacterial liquid is transferred to a BMGY culture medium (500 mL conical flask) containing 50mL at the inoculation proportion of 1% (V/V), and is cultured for 16-18 h at 30 ℃ and 250 rpm; supernatant was removed by centrifugation at 5000rpm, the cells were resuspended in 50ml BMMY medium, and cultured at 30℃at 250rpm, and 0.5ml methanol was added every 24 hours for induction, followed by total induction for 72 hours. After the induction is finished, centrifuging, taking the supernatant, analyzing and measuring, and screening recombinant pichia pastoris strains for efficiently expressing the immunomodulatory peptide.
EXAMPLE 6 high Density fermentation of recombinant Pichia pastoris for efficient expression of immunomodulatory peptide Y6
(1) Single colonies of the recombinant Pichia pastoris strains screened in example 5 for efficient expression of the immunomodulatory peptide were picked and inoculated into 10ml of YPD medium containing G418, cultured overnight at 30℃at 250 rpm.
(2) The overnight cultured bacterial liquid is transferred to 300ml BMGY culture medium with 1% inoculum size, 30 ℃,250rpm, and cultured for 16-24 hours until OD 600 = 2-4 to prepare first-grade seeds.
(3) The primary seed solution was transferred to a fermenter containing 3L of BSM medium at an inoculum size of 10%, and the initial fermentation temperature was set at 30℃and pH5.0, and the rotational speed was 200rpm, with a ventilation of 2L/min.
(4) Until the glycerol in the medium was completely consumed (about 18-24 h), at which time the fermenter dissolved oxygen was raised to approximately 100%, and 50% (W/V) glycerol was initially fed at a rate of 18ml/h/L, with 12ml of PTM1 trace salt being added per liter of 50% (W/V) glycerol. Glycerol feed time is typically 4 hours or more until the wet cell weight reaches 220g/L.
(5) After the glycerol feeding is finished for 2 hours, methanol is fed, and 12ml of PTM1 trace salt is added per liter of methanol. The methanol feeding speed in the methanol induction stage is controlled to be 3 stages: ① 6 hours before methanol induction, the methanol feeding speed is 3ml/L/h; ② The feeding speed is increased by 20 percent in each hour from the induction of methanol for 6 hours until the feeding speed of the methanol reaches 7.2ml/L/h; ③ The methanol feed rate of 7.2ml/L/h was maintained until the total methanol induction time reached 72h.
(6) 50Ml of fermentation broth was collected every 12h during the methanol induction phase, and the wet cell weight, the fermentation broth OD 600 and the fermentation supernatant protein concentration were measured, and the results are shown in FIG. 4. And (5) methanol induction is carried out for 72 hours to finish fermentation, and fermentation supernatant is collected by centrifugation.
EXAMPLE 7 antibacterial Activity detection of fermentation supernatants
The selected indicator bacteria are common pathogenic bacteria in the growth process of livestock and poultry, and are escherichia coli (ESCHERICHIA COLI) ATCC25922, K88 and K99, salmonella typhimurium (Salmonella Typhimurium) SL1344 and ATCC14028, citrobacter typhimurium (Citrobacter rodentium) DBS100 and staphylococcus aureus (Staphylococcus aureus) ATCC6538, ATCC25923 and CVCC1882 respectively.
After streaking the indicator bacteria 3 times, single colonies were inoculated into a conical flask containing 5mL of LB liquid medium, and cultured at 37℃and 200rpm for 12-18 hours. The cultured strain concentration was adjusted to 1X 10 8CFU/mL～5×108 CFU/mL with LB liquid medium and used as a test strain solution. Diluting the test bacterial liquid with LB solid culture medium cooled to about 55 ℃ according to the volume ratio of 1:100, fully and uniformly mixing, then inverting the test bacterial liquid into a sterile culture dish, lightly shaking the flat plate to uniformly spread the solid culture medium, and solidifying the solid culture medium. The sterile oxford cups were gently placed in the assay plates, respectively, 100 μl of the fermentation supernatant collected in example 6 filtered through a sterile 0.22 μm filter was added, and 100 μl of sterile water was added to the blank, and each treatment was repeated 5 times. The flat plate is placed in a constant temperature incubator at 37 ℃ for culturing for 12-18 hours, taken out and the diameter of the formed inhibition zone is measured. The results of the fermentation supernatant with the inhibition zone are shown in FIG. 5, and the diameter data of the inhibition zone are shown in Table 1. According to the detection result, the Pichia pastoris high-density fermentation supernatant for secreting and expressing the immunoregulatory peptide has strong antibacterial activity on common livestock and poultry pathogenic bacteria.
Table 1 statistics of diameter of pathogenic bacteria inhibition zone of high density fermentation supernatant for common livestock and poultry
EXAMPLE 8 purification preparation of recombinant immunomodulatory peptide Y6
The high-density fermentation supernatant collected in example 6 was purified using a Ni NTA Beads 6FF gravity column. The specific operation steps are as follows: ① Adding 5 Lysis buffers (Lysis Buffer solution) with column volumes into a gravity column for balancing, so that the filler is under the same Buffer system as the target protein, and plays a role in protecting the protein; ② Adding the sample into a balanced gravity column, wherein the retention time of the sample is at least 5min, so that the target protein is ensured to be fully contacted with a medium, and the recovery rate of the target protein is improved; ③ Washing with 10-15 times of Wash Buffer (washing Buffer) to remove nonspecifically adsorbed impurity proteins; ④ Eluting the target protein by using an Elution Buffer with a volume of 5 times that of the column, and collecting the eluent; ⑤ Freeze-drying the collected eluent; ⑥ The lyophilized samples were dissolved for Tris-Tricine-SDS-PAGE analysis and the polypeptide yield in the samples was determined. Tris-Tricine-SDS-PAGE is shown in FIG. 6.
The buffers and formulations required for histidine-tagged protein purification are shown in Table 2.
TABLE 2 buffer and formulation for histidine tag protein purification
EXAMPLE 9 stability assay for purification of recombinant immunomodulatory peptide Y6
The simulated intestinal fluid containing pancreatin and the simulated gastric fluid containing pepsin were mixed and incubated with the recombinant immunomodulatory peptide Y6 purified in example 8 at 37 ℃ for 4 hours, and then the minimal inhibitory concentration of the recombinant immunomodulatory peptide Y6 against the Citrobacter aurantiacus (Citrobacter rodentium) ATCC51459 was determined by a microdilution method. A series of gradient peptide solutions were prepared sequentially by a multiple dilution method using a 96-well plate with 0.01% acetic acid and 0.2% bovine serum albumin as dilutions, and the volume of the solution in each well was 50. Mu.L. Then, 50. Mu.L of the bacterial liquid to be tested (about 10 5 CFU/mL) was added to each well, and the culture medium was LB. Culturing at 37deg.C for 18-24 hr, and measuring optical density at 492nm with enzyme label instrument to determine minimum inhibitory concentration of recombinant immunomodulatory peptide Y6 on bacteria. A measured value of less than 0.1 is considered to be bacteria inhibited. Two replicates per test were repeated three times.
The formulas of simulated intestinal fluid containing pancreatin and simulated gastric fluid containing pepsin are shown in table 3.
TABLE 3 formulation of simulated intestinal fluid containing pancreatin and simulated gastric fluid containing pepsin
TABLE 4 stability of purified recombinant immunomodulatory peptide Y6
From table 4, it can be seen that the recombinant immunomodulatory peptide Y6 is resistant to simulated intestinal fluid containing pancreatin and simulated gastric fluid containing pepsin, and retains its original antimicrobial activity after 4 hours of incubation.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A gene for encoding an immunomodulatory peptide Y6 is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. A biological material comprising the gene of claim 1, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, or an engineering bacterium.
3. A recombinant plasmid comprising the gene of claim 1, wherein the starting vector for the recombinant plasmid is pPIC9K.
4. A method for constructing pichia pastoris for secretion expression of an immunomodulatory peptide Y6, comprising: and (3) carrying out codon optimization on the encoding gene of the immunoregulatory peptide Y6 according to the preference of the genetic code of pichia pastoris, cloning the optimized gene into a pichia pastoris expression vector to construct a recombinant expression plasmid, converting the recombinant expression plasmid into pichia pastoris competent cells, and screening high-efficiency expression strains.
5. The method of claim 4, wherein the expression vector is pPIC9K and the pichia competent cell is GS115.
6. The method according to claim 4 or 5, wherein the nucleotide sequence of the optimized gene is shown in SEQ ID NO. 1.
7. The engineered pichia pastoris constructed according to the method of any one of claims 4-6.
8. Use of an immunomodulatory peptide Y6 or an engineered bacterium according to claim 7 in the manufacture of a broad spectrum antibacterial medicament for the treatment of bacterial infections.
9. The use according to claim 8, wherein the bacteria are selected from the group consisting of escherichia coli (ESCHERICHIA COLI) ATCC25922, K88, K99, salmonella typhimurium (Salmonella Typhimurium) SL1344, ATCC14028, citrobacter typhimurium (Citrobacter rodentium) DBS100, ATCC51459, staphylococcus aureus (Staphylococcus aureus) ATCC6538, ATCC25923, CVCC1882.
10. Use of an immunomodulatory peptide Y6 or an engineering bacterium according to claim 7 in the preparation of a feed additive for preventing or treating diarrhea in an animal, for improving growth performance or immune function in an animal; the feed additive has the function of resisting gastrointestinal digestion degradation.
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