WO2024031921A1 - 一种强化乳铁蛋白表达的毕赤酵母及其构建方法与应用 - Google Patents

一种强化乳铁蛋白表达的毕赤酵母及其构建方法与应用 Download PDF

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WO2024031921A1
WO2024031921A1 PCT/CN2022/143078 CN2022143078W WO2024031921A1 WO 2024031921 A1 WO2024031921 A1 WO 2024031921A1 CN 2022143078 W CN2022143078 W CN 2022143078W WO 2024031921 A1 WO2024031921 A1 WO 2024031921A1
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signal peptide
lactoferrin
pichia pastoris
recombinant
fermentation
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French (fr)
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刘龙
陈坚
吕雪芹
堵国成
吴文扬
李江华
刘延峰
周国霖
崔世修
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江南大学
内蒙古蒙牛乳业（集团）股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of metabolic engineering, and in particular refers to a Pichia pastoris that enhances the expression of lactoferrin and its construction method and application.
  • Lactoferrin also known as lactotransferrin, is an iron-binding protein with a molecular weight of approximately 80kDa and is also an important globulin in milk. Studies have shown that LF not only exists in breast milk, but is also widely distributed in tears, saliva, bile and other secretions. Among them, the content of LF is the highest in milk, and the content of LF in human milk ranks second. The concentration is as high as 1.0-3.0 mg/mL, which is 10 times that of cow milk. LF can not only transport iron ions, but also has biological activities such as antibacterial, anti-inflammatory and regulating the body's immune response.
  • LF In addition to its antibacterial activity, LF also has significant bactericidal activity.
  • the alkaline high-density charge on the surface of LF is easy to bind specifically to certain biological molecules of bacteria, disrupting the function of the cell membrane; on the other hand, LF can specifically bind to the bacterial cell membrane, leading to membrane permeability. Increase or even destroy the lipid bilayer, thus playing a bactericidal effect.
  • the bactericidal active area of LF is mainly located in the N lobe, which is different from the iron ion binding area.
  • the N end of LF relies on its own positive charge to generate static electricity with the negatively charged lipopolysaccharide and teichoic acid on the bacterial cell membrane.
  • LF is considered a new antibacterial and anticancer drug.
  • LF has been added to many commercial products, including infant formula powder, thermotherapy drinks, fermented milk, cosmetics and toothpaste.
  • the diverse health-promoting functions of LF and their wide application in real life have stimulated increasing research interest.
  • lactoferrin is mainly obtained through isolation and purification from bovine colostrum.
  • problems in the extraction process such as cumbersome separation steps and high difficulty in purification.
  • heterologous proteins may cause allergic reactions in the human body.
  • researchers use genetic engineering technology to construct a variety of chassis cells (E. coli, yeast, mammalian cells and plant cells) for the production of lactoferrin.
  • the promoter, signal peptide, and terminator in the chassis cells provide the basis for protein expression. These expression elements determine the expression level of the protein. However, these elements are not necessarily the most suitable elements for the protein. In order to discover more expression elements, researchers use green fluorescent protein as an indicator to explore potential expression elements in cells through transcriptome, proteome, and bioinformatics analysis technologies, and identify new expression elements based on changes in fluorescence intensity. .
  • microbial cells Over the past decade, researchers have used microbial cells as hosts to heterologously express human lactoferrin. Although microbial expression systems have the characteristics of fast growth, simple operation, and various post-translational processing and modification functions, there are some inherent defects in the system, such as human-derived protein molecules and cytokines cannot be expressed efficiently in yeast, and the protein products are easy to The formation of multimers leads to protein degradation. The main reason for accumulation is that the synthesized proteins cannot be secreted out of the cell normally, which puts great pressure on the cell secretion system, easily causing cell collapse, leading to the degradation of foreign proteins, and reducing protein synthesis. Yield. At this time, new expression elements need to be discovered to enable the protein to be quickly secreted out of the cell. researchers mainly enhance the expression level of foreign proteins by optimizing the types of signal peptides in the expression system. The expression elements used are often derived from the cells themselves, which limits the secretion of heterologous proteins.
  • the present invention provides a Pichia pastoris that enhances the expression of lactoferrin and its construction method and application.
  • a Pichia pastoris that enhances the expression of lactoferrin and its construction method and application.
  • This method first screens commonly used signal peptides, and then integrates different signal peptides to construct a hybrid signal peptide, which enables lactoferrin to be rapidly transmitted in organelles and promotes the secretion of lactoferrin.
  • the first object of the present invention is to provide a signal peptide, which is obtained by replacing the N-region of the a-factor signal peptide with any one of OST, INU and SUC signal peptides.
  • the a-factor signal peptide sequence is as SEQ. ID NO.11 is shown, and the sequence of the N-region is shown as SEQ ID NO.12;
  • the a-factor signal peptide sequence ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAA GAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCT;
  • sequence of the N-region ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCT.
  • amino acid sequence of the signal peptide INU is shown in SEQ ID NO.2
  • amino acid sequence of the signal peptide OST is shown in SEQ ID NO.1
  • SUC signal peptide The amino acid sequence is shown in SEQ ID NO.3.
  • the amino acid sequence of the INU signal peptide is MKLAYSLLLPLAGVSASVINYKR, see SEQ ID NO. 2.
  • the amino acid sequence of the OST signal peptide is MRQVWFSWIVGLFLCFFNVSSA, see SEQ ID NO.1.
  • amino acid sequence of the SUC signal peptide is MLLQAFLFLLAGFAAKISA, see SEQ ID NO. 3.
  • the second object of the present invention is to provide a gene encoding the signal peptide.
  • the third object of the present invention is to provide a recombinant plasmid carrying the gene.
  • the fourth object of the present invention is to provide a recombinant Pichia pastoris, which includes the signal peptide, the gene or the recombinant plasmid.
  • the INU-a-factor signal peptide sequence is shown in SEQ ID NO.4: atgaagttagcatactccctctttgcttccattggcaggagtcagtgcttcagttatcaattacaagagagctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacag cacaaataacgggttattttgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct;
  • the MEL-a-factor signal peptide sequence is shown in SEQ ID NO.5: atgagagctttcttgtttctcaccgcatgcatcagtttgccaggcgtttttggggtgaacgagactgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttg ccattttccaacagcacacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct;
  • the OST-a-factor signal peptide sequence is shown in SEQ ID NO.6: atgaggcaggtttggttctcttggattgtgggattgttcctatgtttttcaacgtgtcttctgctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgtttt gccaccattttccaacagcacacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct.
  • the PHO-a-factor signal peptide sequence is shown in SEQ ID NO.7: atgttgaagtcagccgtttattcaattttagccgcttctttggttaatgcagctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataac gggttatgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct;
  • SUC-a-factor signal peptide sequence is shown in SEQ ID NO.8: atgcttttgcaagctttccttttcctttttggctggttttgcagccaaaatatctgcagctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccatttt tccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggta tctctcgagaaaagagaggctgaagct.
  • the host strain of the recombinant Pichia pastoris is Pichia pastoris X-33, GS115 or SMD1163.
  • the host strain of the recombinant Pichia pastoris is Pichia pastoris X-33.
  • the recombinant Pichia pastoris vector is selected from piCZa plasmid or piC9k plasmid.
  • the recombinant Pichia pastoris vector is selected from piCZa plasmid.
  • the fifth object of the present invention is to provide a method for constructing the recombinant Pichia pastoris that enhances lactoferrin expression, including the following method: introducing a recombinant expression vector containing a signal peptide into a Pichia pastoris host strain to obtain the recombinant Pichia pastoris Pichia.
  • the sixth object of the present invention is to provide the application of the recombinant Pichia pastoris with enhanced lactoferrin expression in the fermentation production of lactoferrin.
  • the step of fermentation production inoculate the recombinant Pichia pastoris into a culture medium, and after culturing, collect the cells by centrifugation, transfer the cells to the fermentation medium, and supplement Add carbon source and ferment to obtain the lactoferrin.
  • the culture medium is selected from BMGY culture medium.
  • the fermentation medium is selected from BMMY medium.
  • the carbon source is selected from methanol.
  • the culture time is 24-36h.
  • the culture time is 24 hours.
  • the fermentation conditions the fermentation time is: 5-6 days, and the fermentation temperature is 25-30°C.
  • the fermentation time is 5 days.
  • the fermentation temperature is 30°C.
  • the vaccination rate is 3-6%.
  • the vaccination rate is 5%.
  • the components of the culture medium BMGY are: 5g of yeast extract, 10g of tryptone, 350 mL of distilled water, and after steam sterilization at 121°C for 20 minutes, 13.4% YNB (amino acid-free yeast nitrogen) is added.
  • Source Yeast Nitrogen Base
  • Yeast Nitrogen Base 50mL
  • 10% glycerol 50mL
  • 1M pH 6.0 potassium phosphate buffer 50mL
  • biotin 1mL 0.02% biotin 1mL.
  • the components of the fermentation medium BMMY are: 5g of yeast extract, 10g of tryptone, add 350mL of distilled water, and after steam sterilization at 121°C for 20min, add 50mL of 13.4% YNB, 10% 50mL of glycerol, 50mL of 1M potassium phosphate buffer pH 6.0, 1mL of 0.02% biotin.
  • the present invention first further constructs an integrated signal peptide by screening different signal peptides. Through fermentation experiments, it is found that the combination of different signal peptides can improve different protein secretion levels. Among them, when the a-factor signal peptide is integrated with the OST signal peptide, SUC signal peptide, and INU signal peptide, the OST-a-factor integrated signal peptide, SUC-a-factor integrated signal peptide, and INU-a-factor integrated signal peptide are obtained, corresponding to The secretion of lactoferrin of the obtained recombinant strain was significantly improved.
  • the lactoferrin was as high as 98.7mg/L, 84mg/L and 92mg/L respectively. Compared with the lactoferrin production of the strain corresponding to the single signal peptide SUC 4, 3.4 and 3.5 times. Because the yield of SUC single signal peptide lactoferrin is the highest, it was selected as the control experimental group.
  • the present invention can effectively increase the expression of lactoferrin through the combination of different signal peptides.
  • Figure 1 shows the effects of different signal peptides on lactoferrin production in Example 3 of the present invention.
  • Figure 2 shows the effects of the OST-a-factor integrated signal peptide, INU-a-factor integrated signal peptide and SUC-a-factor integrated signal peptide of the present invention on lactoferrin production.
  • Liquid YPD medium 1% yeast extract, 2% peptone, 2% glucose, steam sterilized at 115°C for 20 minutes and then stored at room temperature.
  • BMGY medium 5g of yeast extract, 10g of tryptone, add 350mL of distilled water, steam sterilize at 121°C for 20 minutes, then add 50mL of 13.4% YNB, 50mL of 10% glycerol, and 50mL of 1M pH 6.0 potassium phosphate buffer. 0.02% biotin 1mL.
  • BMMY medium 5g of yeast extract, 10g of tryptone, add 350mL of distilled water, steam sterilize at 121°C for 20 minutes, then add 50mL of 13.4% YNB, 50mL of 10% glycerol, and 50mL of 1M pH 6.0 potassium phosphate buffer. 0.02% biotin 1mL.
  • 1M pH 6.0 potassium phosphate buffer Mix 132mL 1M K 2 HPO 4 and 868mL 1M KH 2 PO 4 , then adjust the pH to 6.0 with potassium hydroxide or phosphoric acid, and steam sterilize at 121°C for 20 minutes.
  • lactoferrin is guided to quickly shuttle in various organelles in Pichia pastoris and be quickly secreted out of the cell to achieve enhanced high-efficiency expression of lactoferrin.
  • the method of the present invention first screens commonly used signal peptides, and then integrates different signal peptides to construct an integrated signal peptide, so that lactoferrin can be rapidly transmitted in organelles and promote the secretion of lactoferrin.
  • piCZa and piC9k plasmids we first replaced the a-factor signal peptide with different signal peptides, and verified the effects of different signal peptides on lactoferrin expression.
  • the present invention provides an integrated signal peptide, which is obtained by replacing the N-region of a-factor signal peptide with any signal peptide of OST, INU and SUC; the amino acid sequence of the integrated signal peptide is such as SEQ ID NO.1 -3 as indicated by any one of them.
  • the present invention provides a gene encoding the integrated signal peptide.
  • the invention provides a recombinant plasmid carrying the gene.
  • the invention provides a recombinant Pichia pastoris, which includes the integrated signal peptide, the gene or the recombinant plasmid.
  • the present invention further provides that the host strain of the recombinant Pichia pastoris is Pichia pastoris X-33, GS115 or SMD1163.
  • the invention provides a method for constructing the recombinant Pichia pastoris with enhanced lactoferrin expression, which includes the following method: inoculating the recombinant Pichia pastoris into the culture medium BMGY, cultivating it for 24h-36h, centrifuging to collect the cells, and The bacterial cells are transferred to the fermentation medium BMMY, carbon sources are added every 24 hours, and the lactoferrin is obtained by fermentation.
  • the present invention not only achieves the purpose of increasing the expression level of lactoferrin, but also provides a method for increasing protein expression elements.
  • the amino acid sequence of the MEL signal peptide is MRAFLFLTACISLPGVFGVNET, see SEQ ID NO.9.
  • the amino acid sequence of the Pho signal peptide is MLKSAVYSILAASLVNA, see SEQ ID NO.10.
  • Collect the cells by centrifugation at 3000 rpm for 5 minutes, wash with sterile water three times, and centrifuge at 3000 rpm for 5 minutes to remove residual glycerol, then resuspend with 25 mL of BMMY culture medium, transfer to a 250 mL Erlenmeyer flask, and incubate at 30°C and 220 rpm for 5 days, 1% methanol was added every 24 hours to allow induction to continue.
  • the fermentation broth was centrifuged to obtain the fermentation supernatant, and the synthesis of lactoferrin was detected by SDS-PAGE. The results showed that the signal peptide can significantly affect the synthesis of lactoferrin.
  • the experimental results are shown in A in Figure 1. WB was further used to verify whether the band was lactoferrin. Ferritin, the experimental results are shown in B in Figure 1.
  • the ELISA kit (Sangon Bioengineering Co., Ltd., Cat. No.: D711315-0096) was used to detect the production of lactoferrin.
  • the signal peptide OST, the signal peptide INU and the signal peptide SUC have a significant promoting effect on the secretion of lactoferrin.
  • the structure of a-factor was analyzed based on structural characteristics and found to be divided into three parts, namely N-region, H-region and C-region.
  • the method for constructing recombinant Pichia pastoris in Example 2 was used to construct a recombinant expression strain, and the primers shown in Table 3 were used to construct integrated signal peptides of different signal peptides and a-factor.
  • the primers used for PCR connection are detailed in Table 3.
  • the correctly sequenced plasmid was linearized and electroporated into Pichia pastoris X-33 using the same method as above to obtain a lactoferrin-expressing strain.
  • the specific fermentation process transfer the correct colonies verified by nucleic acid electrophoresis into 3mL YPD medium, and cultivate overnight at 30°C and 220rpm. The next day, transfer 1 mL of bacterial liquid to a 250 mL Erlenmeyer flask containing 25 mL of BMGY culture medium. Cultivate at 30°C and 220 rpm for about 24 hours until the OD 600 reaches 2-6. Centrifuge at 3000 rpm for 5 minutes to collect the bacteria and wash with sterile water.
  • the OST-a-factor integrated signal peptide, INU-a-factor integrated signal peptide, and SUC-a-factor integrated signal peptide corresponded to the lactoferrin production of the obtained recombinant strain respectively. Reaching 98.7mg/L, 84mg/L and 92mg/L, which are 4, 3.4 and 3.5 times the lactoferrin production of the strain corresponding to the single signal peptide SUC.

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Abstract

本发明公开了一种强化乳铁蛋白表达的毕赤酵母及其构方法与应用。本发明首先筛选了常用的信号肽，然后将不同的信号肽进行整合，构建杂合型信号肽，使乳铁蛋白能够在细胞器中快速传递，促进乳铁蛋白的分泌。本发明通过不同信号肽之间的组合，能够有效的提高乳铁蛋白的表达量，并通过western blot发现乳铁蛋白的表达量增加了约5倍，高达98mg/L。

Description

一种强化乳铁蛋白表达的毕赤酵母及其构建方法与应用 技术领域
本发明属于代谢工程技术领域，尤其是指一种强化乳铁蛋白表达的毕赤酵母及其构方法与应用。
背景技术
乳铁蛋白(Lactoferrin，LF)又称乳转铁蛋白，是一种分子量约为80kDa的铁结合蛋白，也是乳汁中重要的球蛋白。研究表明，LF不仅存在于乳汁中，也广泛分布于泪液、唾液、胆汁等多种分泌液中。其中乳汁中的含量最高，LF在人乳中的含量居第二位，浓度高达1.0-3.0mg/mL，是牛乳中的10倍。LF不仅可以转运铁离子，同时还具有抗菌、消炎及调节机体免疫反应等生物学活性。
除了抑菌活性外LF还具备明显的杀菌活性。一方面，LF表面的碱性高密度电荷易与细菌的某些生物分子进行分特异性结合，扰乱细胞膜的功能；另一方面，LF可以特异性的结合到细菌细胞膜上，导致膜通透性增加，甚至破坏脂质双分子层，从而起到杀菌作用。进一步研究发现，LF的杀菌活性区主要位于N叶，不同于铁离子结合区的区域，LF的N端依赖自身的正电荷与细菌细胞膜上带有负电荷的脂多糖、磷壁酸等产生静电吸引，从而使整个LF结合至细菌的细胞膜上，最终损害细胞膜。因此，LF被认为是一种新型抗菌、抗癌的药物。目前，LF已被添加到许多商业产品中，包括婴儿配方粉、热疗饮料、发酵乳、化妆品和牙膏等。LF的多种促进健康的功能及其在现实生活中的广泛应用激发了越来越多的研究兴趣。
目前，获得乳铁蛋白的方式主要是从牛初乳中通过分离纯化获得。然而，在提取的过程中存在分离步骤繁琐，纯化难度高等问题，重要的是异源蛋白可能引起人体的过敏反应。为了获得大量乳铁蛋白，研究者利用基因工程技 术构建多种底盘细胞(大肠杆菌、酵母、哺乳动物细胞植物细胞)用于生产乳铁蛋白。
底盘细胞中本身的启动子、信号肽、终止子为蛋白的表达提供了基础，这些表达元件决定了蛋白的表达水平，然而这些元件并不一定是最适合蛋白的元件。为了挖掘更多的表达元件，研究者使用绿色荧光蛋白作为指示剂，通过转录组、蛋白质组、以及生物信息分析等技术探索细胞内潜在的表达元件，并根据荧光强度的变化确定新的表达元件。
在过去的十几年中，研究者以微生物细胞作为宿主进行了人乳铁蛋白的异源表达。微生物表达系统虽然具有生长快，操作简单的特点，还具各翻译后加工和修饰功能，但其系统固有的一些缺陷比如人源性蛋白分子、细胞因子不能在酵母中高效表达，并且蛋白产物容易形成多聚体进而导致蛋白降解，积聚的原因主要是合成的蛋白不能正常分泌到胞外，对细胞分泌系统造成很大的压力，易引起细胞的崩溃，导致外源蛋白的降解，减少蛋白的产量。此时需要挖掘新的表达元件使蛋白能够快速的分泌到胞外。研究者主要通过优化表达系统中的信号肽的种类，增强外源蛋白的表达水平，所用的表达元件往往来源于细胞自身，这限制了异源蛋白的分泌。
发明内容
为了解决乳铁蛋白在毕赤酵母中积聚，导致分泌效率低的问题，本发明提供了一种强化乳铁蛋白表达的毕赤酵母及其构建方法与应用。同时为了引导乳铁蛋白在毕赤酵母中快速再各个细胞器中穿梭，并快速的分泌到细胞外，实现增强乳铁蛋白的高效表达。该方法首先筛选了常用的信号肽，然后将不同的信号肽进行整合，构建杂合型信号肽，使乳铁蛋白能够在细胞器中快速传递，促进乳铁蛋白的分泌。具体以piCZa、piC9k质粒出发，首先用不同信号肽将a-factor因子信号肽进行替换，并验证不同信号肽对乳铁蛋白表达量的影响。进一步将上述效果明显的信号肽与a-factor进行整合，构建杂合型信号肽，最后通过将不同信号肽进行组合优化，证明OST-a-factor整合信号肽、SUC-a-factor整合信号肽、INU-a-factor整合信号肽为最佳组合， 获得三株乳铁蛋白高效合成的毕赤酵母菌株。本发明不仅实现提高乳铁蛋白表达量的目的，同时也提供了一种增加蛋白表达元件的方法。
本发明的第一个目的在于提供一种信号肽，所述信号肽由OST、INU和SUC任一信号肽替换a-factor信号肽的N-区域所得，所述a-factor信号肽序列如SEQ ID NO.11所示，所述N-区域的序列如SEQ ID NO.12所示；
在本发明的另一个实施例中，所述a-factor信号肽序列：ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCT；
在本发明的另一个实施例中，所述N-区域的序列：ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCT。
在本发明的另一个实施例中，所述信号肽INU的氨基酸序列如SEQ ID NO.2所示，所述信号肽OST的氨基酸序列如SEQ ID NO.1所示、所述SUC信号肽的氨基酸序列如SEQ ID NO.3所示。
在本发明的另一个实施例中，所述INU信号肽的氨基酸序列为MKLAYSLLLPLAGVSASVINYKR，见SEQ ID NO.2。
在本发明的另一个实施例中，所述OST信号肽的氨基酸序列为MRQVWFSWIVGLFLCFFNVSSA，见SEQ ID NO.1。
在本发明的另一个实施例中，所述SUC信号肽的氨基酸序列为MLLQAFLFLLAGFAAKISA，见SEQ ID NO.3。
本发明的第二个目的在于提供一种基因，所述基因编码所述的信号肽。
本发明的第三个目的在于提供一种重组质粒，所述重组质粒携带所述的 基因。
本发明的第四个目的在于提供一种重组毕赤酵母，所述重组毕赤酵母包括所述信号肽、所述的基因或所述重组质粒。
INU-a-factor信号肽序列如SEQ ID NO.4所示：atgaagttagcatactccctcttgcttccattggcaggagtcagtgcttcagttatcaattacaagagagctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct；
MEL-a-factor信号肽序列如SEQ ID NO.5所示：atgagagctttcttgtttctcaccgcatgcatcagtttgccaggcgtttttggggtgaacgagactgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct；
OST-a-factor信号肽序列如SEQ ID NO.6所示：atgaggcaggtttggttctcttggattgtgggattgttcctatgttttttcaacgtgtcttctgctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct。
PHO-a-factor信号肽序列如SEQ ID NO.7所示：atgttgaagtcagccgtttattcaattttagccgcttctttggttaatgcagctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagct；
SUC-a-factor信号肽序列如SEQ ID NO.8所示：atgcttttgcaagctttccttttccttttggctggttttgcagccaaaatatctgcagctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggta tctctcgagaaaagagaggctgaagct。
在本发明的另一个实施例中，所述重组毕赤酵母的宿主菌为毕赤酵母X-33、GS115或SMD1163。
在本发明的另一个实施例中，所述重组毕赤酵母的宿主菌为毕赤酵母X-33。
在本发明的另一个实施例中，所述重组毕赤酵母的载体选自piCZa质粒或piC9k质粒。
在本发明的另一个实施例中，所述重组毕赤酵母的载体选自piCZa质粒。
本发明的第五个目的在于提供所述的强化乳铁蛋白表达的重组毕赤酵母的构建方法，包括以下方法：将含有信号肽的重组表达载体导入毕赤酵母宿主菌中，得到所述重组毕赤酵母。
本发明的第六个目的在于提供所述的强化乳铁蛋白表达的重组毕赤酵母在发酵生产乳铁蛋白中的应用。
在本发明的另一个实施例中，所述发酵生产的步骤：将所述重组毕赤酵母接种至培养基中，培养之后，离心收集菌体，将菌体转至发酵培养基中，并补加碳源，发酵得到所述乳铁蛋白。
在本发明的另一个实施例中，所述培养基选自BMGY培养基。
在本发明的另一个实施例中，所述发酵培养基选自BMMY培养基。
在本发明的另一个实施例中，所述碳源选自甲醇。
在本发明的另一个实施例中，所述培养时间为24-36h。
在本发明的另一个实施例中，所述培养时间为24h。
在本发明的另一个实施例中，所述发酵条件：发酵时间为：5-6天，发酵温度为25-30℃。
在本发明的另一个实施例中，所述发酵时间为5天。
在本发明的另一个实施例中，发酵温度为30℃。
在本发明的另一个实施例中，所述接种的接种率为3-6％。
在本发明的另一个实施例中，所述接种的接种率为5％。
在本发明的另一个实施例中，所述培养基BMGY的成分：酵母提取物5g，胰蛋白胨10g，加入350mL的蒸馏水，121℃蒸汽灭菌20min后，加入13.4％的YNB(无氨基酸酵母氮源，Yeast Nitrogen Base)50mL，10％的甘油50mL，1M pH 6.0磷酸钾缓冲液50mL，0.02％的生物素1mL。
在本发明的另一个实施例中，所述发酵培养基BMMY的成分：酵母提取物5g，胰蛋白胨10g，加入350mL的蒸馏水，121℃蒸汽灭菌20min后，加入13.4％的YNB 50mL，10％的甘油50mL，1M pH 6.0磷酸钾缓冲液50mL，0.02％的生物素1mL。
本发明的上述技术方案相比现有技术具有以下优点：
1，本发明首先通过筛选不同信号肽，进一步构建整合性信号肽，通过发酵实验发现，不同信号肽间的组合能够提升不同的蛋白分泌水平。其中当a-factor信号肽与OST信号肽、SUC信号肽、INU信号肽进行整合得到OST-a-factor整合信号肽、SUC-a-factor整合信号肽、INU-a-factor整合信号肽，对应所得重组菌株的乳铁蛋白的分泌得到明显的改善，通过western blot发现乳铁蛋白分别高达98.7mg/L、84mg/L和92mg/L，相对于单一信号肽SUC对应菌株的乳铁蛋白的产量的4、3.4和3.5倍，因SUC单一信号肽乳铁蛋白的产量是最高的，因此选用其作为对照实验组。
2，本发明通过不同信号肽之间的组合，能够有效的提高乳铁蛋白的表达量。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中
图1是本发明实施例3中不同信号肽对乳铁蛋白产量的影响。
图2是本发明OST-a-factor整合信号肽、INU-a-factor整合信号肽、SUC-a-factor整合信号肽对乳铁蛋白产量的影响。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明所用试剂和培养基信息：
1，液体YPD培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖，115℃蒸汽灭菌20min后室温保存。
2，BMGY培养基：酵母提取物5g，胰蛋白胨10g，加入350mL的蒸馏水，121℃蒸汽灭菌20min后，加入13.4％的YNB 50mL，10％的甘油50mL，1M pH 6.0磷酸钾缓冲液50mL，0.02％的生物素1mL。
3，BMMY培养基：酵母提取物5g，胰蛋白胨10g，加入350mL的蒸馏水，121℃蒸汽灭菌20min后，加入13.4％的YNB 50mL，10％的甘油50mL，1M pH 6.0磷酸钾缓冲液50mL，0.02％的生物素1mL。
4，1M pH 6.0磷酸钾缓冲液：将132mL 1M的K 2HPO 4和868mL 1M的KH 2PO 4混匀，然后用氢氧化钾或者磷酸将pH调到6.0，121℃蒸汽灭菌20min。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、细胞生物学、重组DNA技术及相关领域的常规技术，这些技术在现有文献中已有完善说明。
为了解决乳铁蛋白在毕赤酵母中积聚问题，因此引导乳铁蛋白在毕赤酵母中快速在各个细胞器中穿梭，并快速的分泌到细胞外，实现增强乳铁蛋白的高效表达。本发明的方法首先筛选了常用的信号肽，然后将不同的信号肽进行整合，构建整合型信号肽，使乳铁蛋白能够在细胞器中快速传递，促进乳铁蛋白的分泌。具体以piCZa、piC9k质粒出发，首先用不同信号肽将a-factor 因子信号肽进行替换，并验证不同信号肽对乳铁蛋白表达量的影响。进一步将上述效果明显的信号肽与a-factor进行整合，构建整合型信号肽，最后通过将不同信号肽进行组合优化，证明OST-a-factor整和信号肽、SUC-a-factor整和信号肽、INU-a-factor整合信号肽为最佳组合，获得三株乳铁蛋白高效合成的毕赤酵母菌株。
本发明提供一种整合信号肽，所述整合信号肽由OST、INU和SUC任一信号肽替换a-factor信号肽的N-区域所得；所述整合信号肽的氨基酸序列如SEQ ID NO.1-3任一项所示。
本发明提供一种基因，所述基因编码所述的整合信号肽。
本发明提供一种重组质粒，所述重组质粒携带所述的基因。
本发明提供一种重组毕赤酵母，所述重组毕赤酵母包括所述整合信号肽、所述的基因或中所述重组质粒。
本发明进一步提供所述重组毕赤酵母的宿主菌为毕赤酵母X-33、GS115或SMD1163。
本发明提供所述的强化乳铁蛋白表达的重组毕赤酵母的构建方法，包括以下方法：将所述重组毕赤酵母接种至培养基BMGY中，培养24h-36h之后，离心收集菌体，将菌体转至发酵培养基BMMY中，每24小时补加碳源，发酵得到所述乳铁蛋白。
本发明不仅实现提高乳铁蛋白表达量的目的，同时也提供了一种增加蛋白表达元件的方法。
实施例1信号肽的扩增
以酵母P.pastoris X-33基因组为模板，利用表1所列的引物序列分别扩增对应的信号肽(INU信号肽(序列见SEQ ID NO.2、MEL信号肽(序列见SEQ ID NO.9)、OST信号肽(序列见SEQ ID NO.1、Suc信号肽(序列见SEQ ID NO.3、Pho信号肽(序列见SEQ ID NO.10)，并将上述得到的PCR扩增产 物消化之后转化大肠杆菌JM109感受态(以picza为载体)，涂布含有博来霉素的平板，获得相应五种转化子。将五种转化子分别过夜培养，提取质粒，然后通过测序获得正确的转化子。
表1扩增信号肽的引物序列
Figure PCTCN2022143078-appb-000001
所述MEL信号肽的氨基酸序列为MRAFLFLTACISLPGVFGVNET，见SEQ ID NO.9。
所述Pho信号肽的氨基酸序列为MLKSAVYSILAASLVNA，见SEQ ID NO.10。
实施例2不同信号肽对乳铁蛋白表达的影响
以上述测序正确的质粒为模板，利用线性化引物(如表2所述)进行扩增，获得线性化片段，通过GeneJET PCR纯化试剂盒(货号：K0702)纯化线性化片段。取80μL毕赤酵母X-33感受态与10μL线性化重组质粒DNA混合均匀，并转移到-20℃条件下冰浴过的0.2cm电转化杯中，并将装有混合液的电转化杯冰浴5min；调整好电转化仪的参数，置于毕赤酵母档，电压1.5千伏，电容25微法，电阻200欧姆，时间约为5毫秒；电击后，迅速向转化杯中加入1mL冰上预冷的1M山梨醇溶液，轻轻吹吸混匀，将混合液吸出，并转移到离心管中；30℃静置培养1-2h，3000rpm离心5min，弃800μL上清，剩余菌体吹匀，涂布于YPDS培养基(含有100μg/mL的博来霉素)平板上或MD平板上，30℃恒温培养箱中培养2-4天，直长出菌落，通过菌落PCR验证菌落是否正确。
表2线性化引物
Figure PCTCN2022143078-appb-000002
将正确的菌落转接到3mL YPD培养基中，30℃，220rpm过夜培养。次日吸取1mL菌液转接到有25mL BMGY培养基的250mL锥形瓶中，30℃，220rpm培养，使OD 600达到2-6，大约培养16-18h。3000rpm离心5min收集菌体，无菌水清洗3次，离心条件为3000rpm离心5min，以去除残余的甘油，然后用25mL BMMY培养基重悬，转入250mL锥形瓶中，30℃，220rpm培养5天，每24小时补加1％的甲醇，使诱导持续发生。
发酵液离心获得发酵上清，通过SDS-PAGE检测乳铁蛋白的合成，结果显示信号肽能够明显影响乳铁蛋白的合成，实验结果见图1中的A，进一步利用WB验证条带是否为乳铁蛋白，实验结果见图1中的B。利用ELISA试剂盒(生工生物工程股份有限公司，货号：D711315-0096)检测乳铁蛋白的 产量，结果显示，其中影响最明显的信号肽是OST、INU和SUC，乳铁蛋白的产量分别达到24.3mg/L、22.8mg/L和25.6mg/L，实验结果见图1中的C。
实施例3整合信号肽的构建
上述结果已知信号肽OST、信号肽INU和信号肽SUC对乳铁蛋白的分泌具有明显的促进作用。为进一步探索不同信号肽对乳铁蛋白产量的影响，根据结构特征分析a-factor的结构，发现可以分为三个部分，分别是N-区域、H-区域和C-区域，实验结果见图2A，利用PCR将信号肽OST、信号肽INU和信号肽SUC代替N-区域，从而构建整合型信号肽，得到OST-a-factor整合信号肽、INU-a-factor整合信号肽、SUC-a-factor整合信号肽。
利用实施例2中构建重组毕赤酵母的方法构建重组表达菌株，用表3所示的引物构建不同信号肽与a-factor的整合信号肽，PCR连接所用引物详见表3。
表3.扩增杂整合信号肽的引物序列
Figure PCTCN2022143078-appb-000003
Figure PCTCN2022143078-appb-000004
将上述获得的产物经过DPNI消化之后，向大肠杆菌JM109感受态中加入10μL消化后的PCR产物，轻轻吹吸混匀，冰浴30min。将EP管置于浮漂上，放入42℃的水浴锅中热激90s后，立即放在冰上，冰浴1-2min。在超净台上，每管加入800μL的LB培养基，37℃，220rpm孵育1h左右，使细胞复苏。4000rpm离心2min，去掉大部分上清，留下大约100μL的上清重悬菌体，在超净台上涂布于带有Zeocin抗的LB固体培养基上，在37℃恒温培养箱里过夜培养。
将测序正确的质粒进行线性化，利用同上的方法电转化毕赤酵母X-33，获得表达乳铁蛋白菌株。
并利用同样的发酵方式对表达乳铁蛋白重组菌株进行发酵，具体的发酵过程：将通过核酸电泳验证正确的菌落转接到3mL YPD培养基中，30℃，220rpm过夜培养。次日吸取1mL菌液转接到有25mL BMGY培养基的250mL锥形瓶中，30℃，220rpm培养，大约培养24h，使OD 600达到2-6。3000rpm离心5min收集菌体，无菌水清洗3次，离心条件3000rpm离心5min，以去除残余的甘油，然后用25mL BMMY培养基重悬，转入250mL锥形瓶中，30℃，220rpm培养5天，每24小时补加1％的甲醇，使诱导持续发生。图2中B的SDS-PAGE显示，相比较a-factor信号肽，OST-a-factor整合信号肽、INU-a-factor整合信号肽、SUC-a-factor整合信号肽能够改善乳铁蛋白的分泌。实验结果见图2中C，通过ELISA检测发现，OST-a-factor整合信号肽、INU-a-factor整合信号肽、SUC-a-factor整合信号肽对应所得重组菌株的乳铁蛋白的产量分别达到98.7mg/L、84mg/L和92mg/L，是单一信号肽SUC对应菌株的乳铁蛋白的产量的4、3.4和3.5倍。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种信号肽，其特征在于，所述信号肽由OST、INU和SUC任一信号肽替换a-factor信号肽的N-区域所得，所述a-factor信号肽序列如SEQ ID NO.11所示，所述N-区域的序列如SEQ ID NO.12所示。
  2. 根据权利要求1所述的信号肽，其特征在于，所述信号肽INU的氨基酸序列如SEQ ID NO.2所示，所述信号肽OST的氨基酸序列如SEQ ID NO.1所示、和所述SUC信号肽的氨基酸序列如SEQ ID NO.3所示。
  3. 一种基因，其特征在于，所述基因编码权利要求1或2所述的信号肽。
  4. 一种重组质粒，其特征在于，所述重组质粒携带权利要求3所述的基因。
  5. 一种重组毕赤酵母，其特征在于，所述重组毕赤酵母包括权利要求1或2中所述信号肽、权利要求3所述的基因或权利要求4中所述重组质粒。
  6. 根据权利要求5所述的强化乳铁蛋白表达的重组毕赤酵母，其特征在于，所述重组毕赤酵母的宿主菌为毕赤酵母X-33、GS115或SMD1163。
  7. 一种权利要求6所述的强化乳铁蛋白表达的重组毕赤酵母的构建方法，其特征在于，包括以下方法：将含有信号肽的重组表达载体导入毕赤酵母宿主菌中，得到所述重组毕赤酵母。
  8. 权利要求7所述的强化乳铁蛋白表达的重组毕赤酵母在发酵生产乳铁蛋白中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述发酵生产的步骤：将所述重组毕赤酵母接种至培养基中，培养24-36 h之后，离心收集菌体，将菌体转至发酵培养基中，并补加碳源，发酵得到所述乳铁蛋白；所述接种的接种率为3-6％。
  10. 根据权利要求9所述的应用，其特征在于，所述发酵条件：发酵时间为：5-6天，发酵温度为25-30℃。
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