An engineered heterologous recombinant pichia pastoris for producing anti-tumor ginseng peptide and application thereof

Abstract

The application discloses an anti-tumor ginseng peptide producing heterologous recombinant Pichia pastoris engineering bacteria, the engineering bacteria are preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC NO:29803, and the preservation date is January 29, 2024. The engineering bacteria are integrated with a FKEHGY polypeptide gene segment with anti-colon cancer activity derived from fresh ginseng leaves by a gene means, the FKEHGY polypeptide gene segment is enhanced in activity by 30 times of repetition, and a molecular chaperone protein HAC1 is expressed simultaneously, the homologous characteristics of HAC1 and Atf / CREB1 are fully utilized, protein correct folding and secretion are helped, and the engineering bacteria have the characteristics of stably and efficiently producing recombinant anti-tumor ginseng peptide F0630.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a heterologous recombinant Pichia pastoris engineering bacterium for producing an anti-tumor ginseng peptide and application thereof. BACKGROUND

[0002] It is particularly important to research and develop new anti-tumor drugs, especially those with low toxicity and high efficacy. Among numerous anti-tumor drugs, polypeptide drugs have attracted much attention due to their unique advantages. These drugs have a small molecular weight, which allows them to penetrate cell membranes more effectively, thereby improving their bioavailability. At the same time, they have strong targeting ability, which enables them to act more precisely on tumor cells and reduce damage to normal cells. In addition, polypeptide drugs have relatively low toxicity, which makes them have fewer side effects on patients during treatment, thereby improving the quality of life and safety of patients. Therefore, polypeptide drugs have great potential and application prospects in anti-tumor treatment, and are an important direction for current and future research on anti-tumor drugs.

[0003] The use of ginseng has a history of more than 2000 years, and people believe that ginseng has the effect of prolonging life. The name "Panax" in the genus name of ginseng comes from Greek, meaning long life and curing all diseases, and its application history can be traced back thousands of years. In addition, ginseng peptides have anti-tumor, anti-stress and antioxidant activities. The preparation of ginseng peptides can be achieved by three methods: isolation and extraction from nature, artificial chemical synthesis, and biological recombinant expression. The isolation and extraction method is limited by the complexity of operation, time consumption, high cost and low extraction efficiency. In contrast, the chemical synthesis method can produce high-purity ginseng peptides, but it is limited by high cost, many by-products, complicated separation and purification process, and possible environmental impact. The biological recombinant expression technology stands out with its shorter culture period, low cost and high expression amount, and has become a research hotspot. This method expresses the gene of ginseng peptide in a host organism, which not only improves the production efficiency, but also reduces the environmental burden, indicating the future development direction of ginseng peptide production.

[0004] Pichia pastoris is a single-celled eukaryote. It has the characteristics of prokaryotes, such as easy cultivation, fast reproduction and convenient genetic engineering operation, and also has the ability of eukaryotes to correctly fold and post-translational processing and modification of gene products. Therefore, Pichia pastoris is often used to express foreign proteins. Therefore, based on synthetic biology technology, using Pichia pastoris as a recombinant protein expression system to develop a recombinant strain that can efficiently express functional ginseng peptides and their derivatives shows greater application prospects. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide and an application thereof to solve the problems of the prior art.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is: a heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide, characterized in that the engineering bacteria is Pichia pastoris, which is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO: 29803 and the preservation date of January 29, 2024.

[0007] The heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide is characterized in that the host bacteria of the engineering bacteria is Pichia pastoris GS115, and the host bacteria has the recombinant plasmid pPIC9K-F0630 linearized by Sal I enzyme and the recombinant plasmid pGAPZB-HAC1 linearized by Avr II enzyme.

[0008] The heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide is characterized in that the recombinant plasmid pPIC9K-F0630 is a recombinant plasmid pPIC9K-F0630 obtained by inserting the gene encoding the anti-tumor ginseng peptide into the pPIC9K plasmid.

[0009] The heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide is characterized in that the nucleic acid sequence of the anti-tumor ginseng peptide is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0010] The heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide is characterized in that the recombinant plasmid pGAPZB-HAC1 is a recombinant plasmid pGAPZB-HAC1 obtained by inserting the gene encoding the molecular chaperone protein HAC1 into the pGAPZB plasmid.

[0011] The heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptide is characterized in that the nucleotide sequence of the molecular chaperone protein HAC1 is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0012] The application also provides a method for producing the anti-tumor ginseng peptide, characterized by comprising: fermenting and inducing the recombinant Pichia pastoris engineering bacteria.

[0013] The method, characterized in that the fermenting and inducing comprises: streaking culture of the recombinant Pichia pastoris engineering bacteria on a YPD culture medium to obtain single colonies, picking the single colonies and transferring into a BMGY flask culture medium for culture until OD 600 = 10-15, resuspending the bacterial bodies in a BMMY flask culture medium containing methanol, and performing 72h of induction fermentation culture under the condition of 28-30 DEG C and 200rpm rotation speed, wherein methanol is supplemented every 24h during the induction fermentation culture process, and centrifugation is performed to obtain the anti-tumor ginseng peptide.

[0014] The method, characterized in that the amount of the methanol supplemented every 24h during the induction fermentation culture process is 1% (w / v) of the final concentration of the system.

[0015] The method, characterized in that the OD600 of the system obtained by resuspending the bacterial bodies in the BMMY flask culture medium containing methanol is 1.

[0016] Compared with the prior art, the application has the following advantages:

[0017] 1. In the application, the Pichia pastoris GS115 is used as the host bacteria, and the anti-tumor ginseng peptide is obtained by co-expressing the chaperone protein and the anti-tumor ginseng peptide, so that the ginseng peptide capable of effectively inhibiting the growth of tumor cells is obtained, and the synthesis of the anti-tumor ginseng peptide by the microbial method is realized.

[0018] 2. The method for producing the anti-tumor ginseng peptide by using the heterologous recombinant Pichia pastoris engineering bacteria comprises fermentation by using methanol as the substrate, and has the characteristics of stable product and low fermentation cost.

[0019] 3. The heterologous recombinant Pichia pastoris engineering bacteria for producing the anti-tumor ginseng peptide has great potential in the preparation of the anti-tumor ginseng peptide and the products containing the anti-tumor ginseng peptide, and has a wide application prospect.

[0020] The technical solutions of the application will be further described in detail below in combination with the drawings and the embodiments.

[0021] Drawings of the specification

[0022] Figure 1 It is a recombinant plasmid pPIC9K-F0630 plasmid map;

[0023] Figure 2 It is a molecular chaperone constitutive expression plasmid pGAPZB-HAC1 plasmid map;

[0024] Figure 3Figure 1 is a linearized pPIC9K-F0630 gel electrophoresis chart;

[0025] Figure 4 Figure 2 is a GS115-pPIC9K-F0630 colony PCR gel electrophoresis chart;

[0026] Figure 5 Figure 3 is a SDS-PAGE chart of recombinant ginseng peptide F0630;

[0027] Figure 6 Figure 4 is a chart of the inhibition rate of ginseng peptides with different numbers of sequence FKEHGY repeats on CT-26 cells. DETAILED DESCRIPTION

[0028] In the following examples, various processes and methods not described in detail are conventional methods known in the art.

[0029] Example 1

[0030] Construction of recombinant Pichia pastoris expression vector pPIC9K and codon optimization of ginseng peptide

[0031] (1) The ginseng peptide gene F0630 is codon optimized according to the codon bias of Pichia pastoris, EcoR I, Not I and AAAAGA signal peptide cleavage sites are introduced, pPIC9K is used as an expression vector to construct the recombinant plasmid pPIC9K-F0630; the optimized nucleic acid sequence is shown in SEQ. ID. No. 1, and the amino acid sequence is shown in SEQ ID NO. 2; the insertion site of the optimized gene sequence is after the AAAAGA signal peptide cleavage site; the recombinant plasmid pPIC9K-F0630 is obtained and submitted to China GenScript Biotech Co., Ltd.; the ginseng hexapeptide has good antitumor efficacy, but its content in ginseng is very low, and it cannot be prepared on a large scale by plant extraction, and it is difficult to be expressed efficiently by genetic engineering due to the short coding gene. The present application uses genetic engineering technology to repeat the expression gene of ginseng hexapeptide 30 times, optimizes it according to the codon bias of Pichia pastoris, and translates and expresses the derived ginseng peptide F0630 with antitumor synergistic effect; the plasmid map of the recombinant plasmid pPIC9K-F0630 is shown in Figure 1 ;

[0032] SEQ ID NO. 1:

[0033] TTCAAAGAGCACGGCTACTTTAAGGAACATGGCTACTTCAAGGAACACGGATACTTTAAAGAACATGGATACTTTAAAGAGCATGGATACTTCAAAGAACACGGTTACTTCAAAGAGCATGGTTACTTCAAAGAGCATGGTTATTTCAAAGAGCACGGTTACTTTAAAGAGCACGGTTACTTCAAAGAGCACGGATACTTCAAAGAACATGGCTATTTCAAAGAGCATGGCTACTTTAAAGAACACGGATATTTCAAGGAGCATGGCTATTTCAAAGAGCATGGATATTTCAAAGAACACGGCTACTTCAAAGAGCACGGTTATTTCAAAGAGCACGGCTATTTCAAAGAACATGGTTACTTCAAAGAGCACGGCTACTTTAAGGAACATGGCTACTTCAAGGAACACGGATACTTTAAAGAACATGGATACTTTAAAGAGCATGGATACTTCAAAGAACACGGTTACTTCAAAGAGCATGGTTACTTCAAAGAGCATGGTTATTTCAAAGAGCACGGTTACTTTAAAGAGCACGGTTAC

[0034] SEQ ID NO. 2:

[0035] FKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGYFKEHGY

[0036] (2) using Sal I to single-enzyme cut the recombinant plasmid pPIC9K-F0630 to obtain a linearized recombinant plasmid, and after the linearized recombinant plasmid is purified by alcohol precipitation, the linearized recombinant plasmid is electroporated into Pichia pastoris engineering bacteria GS115 competent cells, positive transformant screening is performed on a histidine-deficient MD plate, high copy screening is performed on a YPD plate with G418 resistance, and a template transformant is obtained, and the template transformant is subjected to PCR identification to obtain the recombinant Pichia pastoris engineering bacteria GS115 / pPIC9K-F0630; the mass of the recombinant plasmid pPIC9K-F0630 is 10 μg; the electroporation is performed at a voltage of 1.5 kV by using a 2-mm electroporation cup; the histidine-deficient MD plate is an MD solid culture medium sterilized at 121 ℃ for 20 min, and the MD solid culture medium comprises 1.34% (w / v) YNB, 2% (w / v) glucose, 4×10 -5 Figure 3 Figure 4

[0037] The primers in the PCR identification are 3’ACAACTAATTATTCGAAGGATCCAAACGAT and 5’GTGCCTGACTGCGTTAGCAATTTAACT.

[0038] Example 2

[0039] The chaperone gene HAC1 is constructed in the plasmid pGAPZB, and the specific embodiment is as follows:

[0040] (1) 0.5 μL of GS115 genomic DNA is used as an amplification template, chaperone gene HAC1 amplification primers are added, Primer Star Max high-fidelity enzyme amplification is performed, the amplification product is detected and confirmed by agarose gel electrophoresis, and then the product is recovered by gel, and EcoRI and Xho I are used for enzyme cutting; the nucleic acid sequence of the chaperone gene HAC1 is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; the PCR amplification system and amplification conditions are shown in Table 1-1; ​​​

[0041] The amplification primers are:

[0042] HAC1-F: GAATTCATGCCCGTAGATTCTTCTCATAA;

[0043] HAC1-R: CTCGAGTCACCTGATCGCTATGCATGTCAACT;

[0044] SEQ ID NO. 3:

[0045] ATGCCCGTAGATTCTTCTCATAAGACAGCTAGCCCACTTCCACCTCGTAAAAGAGCAAAGACGGAAGAAGAAAAGGAGCAGCGTCGAGTGGAACGTATCCTACGTAATAGGAGAGCGGCCCATGCTTCCAGAGAGAAGAAACGAAGACACGTTGAATTTCTGGAAAACCACGTCGTCGACCTGGAATCTGCACTTCAAGAATCAGCCAAAGCCACTAACAAGTTGAAAGAAATACAAGATATCATTGTTTCAAGGTTGGAAGCCTTAGGTGGTACCGTCTCAGATTTGGATTTAACAGTTCCGGAAGTCGATTTTCCCAAATCTTCTGATTTGGAACCCATGTCTGATCTCTCAACTTCTTCGAAATCGGAGAAAGCATCTACATCCACTCGCAGATCTTTGACTGAGGATCTGGACGAAGATGACGTCGCTGAATATGACGACGAAGAAGAGGACGAAGAGTTACCCAGGAAAATGAAAGTCTTAAACGACAAAAACAAGAGCACATCTATCAAGCAGGAGAAGTTGAATGAACTTCCATCTCCTTTGTCATCCGATTTTTCAGACGTAGATGAAGAAAAGTCAACTCTCACACATTTAAAGTTGCAACAGCAACAACAACAACCAGTAGACAATTATGTTTCTACTCCTTTGAGTCTTCCGGAGGATTCAGTTGATTTTATTAACCCAGGTAACTTAAAAATAGAGTCCGATGAGAACTTCTTGTTGAGTTCAAATACTTTACAAATAAAACACGAAAATGACACCGACTACATTACTACAGCTCCATCAGGTTCCATCAATGATTTTTTTAATTCTTATGACATTAGCGAGTCGAATCGGTTGCATCATCCAGCAGTGATGACGGATTCATCTTTACACATTACAGCAGGCTCCATCGGCTTTTTCTCTTTGATTGGGGGGGGGGAAAGTTCTGTAGCAGGGAGGCGCAGTTCAGTTGGCACATATCAGTTGACATGCATAGCGATCAGGTGA

[0046] SEQ ID NO.4:

[0047] MPVDSSHKTASPLPPRKRAKTEEEKEQRRVERILRNRRAAHASREKKRRHVEFLENHVVDLESALQESAKATNKLKEIQDIIVSRLEALGGTVSDLDLTVPEVDFPKSSDLEPMSDLSTSSKSEKASTSTRRSLTEDLDEDDVAEYDDEEEDEELPRKMKVLNDK NKSTSIKQEKLNELPSPLSSDFSDVDEEKSTLTHLKLQQQQQPVDNYVSTPLSLPEDSVDFINPGNLKIESDENFLLSSNTLQIKHENDTDYITTAPSGSINDFFNSYDISESNRLHHPAVMTDSSLHITAGSIGFFSLIGGGESSVAGRRSSVGTYQLTCIAIR

[0048] (2) Plasmid pGAPZB was digested with the same EcoRI and Xho I enzymes, recovered by column chromatography, and the amplification product was ligated under the GAP promoter to construct the molecular chaperone constitutive expression plasmid pGAPZB-HAC1; the amplification product was located after the EcoRI restriction site; the plasmid diagram of the molecular chaperone constitutive expression plasmid pGAPZB-HAC1 is shown below. Figure 2 As shown;

[0049] (3) 10 μg of the molecular chaperone constitutive expression plasmid was digested with Avr II single enzyme to obtain a linearized recombinant plasmid. After purification by alcohol precipitation, it was electroporated into the GS115-pPIC9K-F0630 to obtain a recombinant strain capable of producing antitumor ginseng peptide.

[0050] Table 1-1 PCR amplification system

[0051]

[0052] Molecular chaperones are primarily responsible for the proper folding of other proteins and preventing their aggregation, ensuring normal protein function. They also participate in protein transport, repair, and degradation, maintaining intracellular protein homeostasis. In this invention, the molecular chaperone gene HAC1, which is homologous to Atf / CREB1, is co-expressed with the target gene to regulate the unfolded protein response, significantly increasing the yield of the target protein.

[0053] Example 3: Production of antitumor ginseng peptides by shake-flask fermentation

[0054] (1) The recombinant strain capable of producing anti-tumor ginseng peptide obtained in Example 2 was streaked on YPD medium to obtain single colonies; the YPD medium was obtained by sterilizing YPD solid medium at 115℃ for 20 min, and the YPD solid medium included: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose and 2% (w / v) agar powder;

[0055] (2) Pick a single colony and transfer it into 10 mL of BMGY shake flask medium. Incubate at 30°C and 200 rpm until OD500. 600 =10-15, centrifuged at 8000 rpm for 10 min at 4℃ to remove the supernatant and collect the bacterial cells; the BMGY shake flask culture medium includes: 1% (w / v) yeast extract, 2% (w / v) peptone, 10% (w / v) 1M phosphate buffer with pH 6.0, 1.34% (w / v) YNB and 1% (w / v) glycerol; wherein the preparation method of the 1M phosphate buffer with pH 6.0 includes: mixing 11.484g K2HPO4 and 59.024g KH2PO4 with water and making up to 500mL to obtain the 1M phosphate buffer with pH 6.0;

[0056] (3) According to the initial OD 600 1. The bacterial culture was suspended in BMMY shake flask medium containing 1% (w / v) methanol and induced to ferment at 30°C and 200 rpm. During the induced fermentation process, methanol was added every 24 hours to a final concentration of 1% (w / v). After 72 hours, the culture was centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was then subjected to SDS-PAGE protein electrophoresis to confirm the bands, and the ginseng peptides were collected. In this invention, the suitable temperature for induced fermentation is 28°C to 30°C. 0℃, in this embodiment the temperature is 30℃; the BMMY shake flask medium containing 1% (w / v) methanol is the BMMY shake flask medium containing 1% (w / v) methanol obtained by adding methanol to BMMY medium, wherein the methanol content is 1% (w / v); the BMMY medium includes 1% (w / v) yeast extract, 2% (w / v) peptone, 10% (w / v) phosphate buffer with a concentration of 1M and a pH of 6.0 and 1.34% (w / v) YNB;

[0057] like Figure 5 As shown, the SDS-PAGE protein electrophoresis results show obvious specific bands, indicating that the method of the present invention successfully obtained ginseng peptides.

[0058] Example 4: Experiment on the inhibition of tumor cells by anti-tumor ginseng peptides

[0059] 100 μL per well, 1 × 10 4 To determine the optimal cell volume, CT26 cells were seeded into 96-well plates and cultured overnight in a CO2 incubator. Following this, the cells were treated with ginseng peptide and cultured for another 48 hours. Then, 10 μL of 5 mg / ml MTT reagent was added to each well, and the cells were incubated for 4 hours. Finally, 100 μL of DMSO was added, and the absorbance at 570 nm was measured using a microplate reader. The overnight culture conditions were: saturated humidity, 37°C, and 5% CO2. The blank group consisted of normal cell culture medium, while the experimental groups contained different concentrations of ginseng peptide. The concentrations and corresponding experimental results are shown below. Figure 6 As shown, (FKEHGY)30 is the anti-tumor ginseng peptide of the present invention, and FKEHGY is the original ginseng hexapeptide without fragment duplication, which was purchased from Sangon Biotech.

[0060] Inhibition rate (%) = (OD value of control group - OD value of experimental group) / OD value of control group

[0061] like Figure 6 By applying chemically synthesized monomeric ginseng peptide and 30-repeated antitumor ginseng peptide at different concentrations to CT26 cells, the results showed that the recombinant ginseng peptide F0630 obtained by repeating the selected sequence FKEHGY 30 times had a significant inhibitory effect on CT26 cells.

[0062] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A heterologous recombinant Pichia pastoris engineering bacteria for producing anti-tumor ginseng peptides, characterized in that, The engineering bacteria are Pichia pastoris, deposited in the China General Microbiological Culture Collection Center on January 29, 2024, with the accession number CGMCC NO: 29803; the host bacteria of the engineering bacteria are Pichia pastoris GS115, and the host bacteria have the recombinant plasmid pPIC9K-F0630 linearized by Sal I enzyme and the recombinant plasmid pGAPZB-HAC1 linearized by Avr II enzyme; the recombinant plasmid pPIC9K-F0630 is a recombinant plasmid pPIC9K-F0630 obtained by inserting a gene encoding an anti-tumor ginseng peptide into a pPIC9K plasmid; the nucleic acid sequence of the anti-tumor ginseng peptide is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the recombinant plasmid pGAPZB-HAC1 is a recombinant plasmid pGAPZB-HAC1 obtained by inserting a gene encoding a chaperone protein HAC1 into a pGAPZB plasmid; the nucleotide sequence of the chaperone protein HAC1 is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO.

4.

2. A method for producing an anti-tumor ginseng peptide, characterized by, The recombinant Pichia pastoris engineering bacteria of claim 1 are subjected to fermentation induction; the fermentation induction comprises: streaking culture of the recombinant Pichia pastoris engineering bacteria on a YPD culture medium to obtain single colonies, picking the single colonies and transferring them into a BMGY flask culture medium for culture until OD600 = 10-15, obtaining bacterial bodies, resuspending the bacterial bodies in a BMMY flask culture medium containing methanol, and performing 72 h of induction fermentation culture at 28-30 DEG C and a rotation speed of 200 rpm; methanol is added every 24 h during the induction fermentation culture process, centrifugation is performed, and an anti-tumor ginseng peptide is obtained; the amount of methanol added every 24 h during the induction fermentation culture process is 1% (w / v) of the final concentration of the system; the OD600 of the bacterial bodies resuspended in the BMMY flask culture medium containing methanol is 1. ​

Images