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CN-121975648-A - Pichia pastoris gene engineering strain GGM4 and construction method and application thereof

CN121975648ACN 121975648 ACN121975648 ACN 121975648ACN-121975648-A

Abstract

The invention discloses a Pichia pastoris gene engineering strain for expressing recombinant human granulocyte-macrophage colony stimulating factor, a construction method and application thereof, wherein the classification of the Pichia pastoris gene engineering strain GGM4 is named as Pichia pastoris (syn.Komagataella phaffii), and the preservation number is CGMCC No.39117. The engineering strain is obtained by combining and using the Pichia pastoris endogenous secretion signal peptide PRY2 and the Saccharomyces cerevisiae secretion signal peptide alpha MF and over-expressing Pichia pastoris endogenous genes PDI1, SSA4 and SNC2, thereby realizing the high-efficiency expression of recombinant human granulocyte-macrophage colony stimulating factor. After the strain is subjected to high-density fermentation in a 3L fermentation tank, the titer of recombinant human granulocyte-macrophage colony stimulating factor in fermentation supernatant is more than 4 g/L. The strain can express high-titer recombinant human granulocyte-macrophage colony stimulating factor and has wide industrial application value in the field of recombinant protein drug production.

Inventors

  • GUAN WENJUN
  • CHENG ZHENZHEN
  • SONG YUCHAO

Assignees

  • 浙江大学

Dates

Publication Date
20260505
Application Date
20260210

Claims (8)

  1. 1. A pichia pastoris gene engineering strain GGM4 for expressing recombinant human granulocyte-macrophage colony stimulating factor is characterized in that the pichia pastoris gene engineering strain GGM4 takes pichia pastoris GS115 strain as host strain to express the recombinant human granulocyte-macrophage colony stimulating factor, and the nucleotide sequence of the recombinant human granulocyte-macrophage colony stimulating factor is shown as SEQ ID NO. 1.
  2. 2. The Pichia pastoris gene engineering strain GGM4 according to claim 1, wherein the Pichia pastoris is designated by Pichia pastoris (syn. Komagataellaphaffii) and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 39117 and the preservation date of 2026, 01 and 04.
  3. 3. A method for constructing a pichia pastoris gene engineering strain GGM4 according to any one of claims 1 to 2, comprising the steps of: (1) Constructing plasmid pPIC9-PRY2-hCSF2 containing chimeric gene PRY2-hCSF2 by using gene cloning and recombination method, wherein the nucleotide sequence of the chimeric gene PRY2-hCSF2 is shown as SEQ ID NO. 2; (2) Integrating plasmid pPIC9-PRY2-hCSF2 on chromosome I of Pichia pastoris GS115 strain by homologous recombination method to obtain single copy strain GGM1; (3) Integrating a second copy of the chimeric gene PRY2-hCSF2 on a chromosome II of a GGM1 strain to obtain a GGM2 strain, integrating a chimeric gene alpha MF-hCSF2 formed by a saccharomyces cerevisiae secretion signal peptide alpha MF sequence and a recombinant human granulocyte-macrophage colony stimulating factor sequence on a chromosome III and a chromosome IV of the GGM2 strain respectively to obtain a signal peptide combined multicopy strain GGM3, wherein the nucleotide sequence of the chimeric gene alpha MF-hCSF formed by the saccharomyces cerevisiae signal peptide alpha MF and the recombinant human granulocyte-macrophage colony stimulating factor is SEQ ID NO. 3; (4) Based on the strain GGM3, the Pichia pastoris gene engineering strain GGM4 is obtained by using a promoter P TEF1 to overexpress the PDI1 gene, using a promoter P SSA4 to overexpress the SSA4 gene and using a promoter P 0510 to overexpress the SNC2 gene.
  4. 4. The method according to claim 3, wherein in the step (4), the nucleotide sequence of the promoter P TEF1 is SEQ ID NO. 4, the nucleotide sequence of the promoter P SSA4 is SEQ ID NO. 5, the nucleotide sequence of the promoter P 0510 is SEQ ID NO. 6, the nucleotide sequence of the gene PDI1 is SEQ ID NO. 7, the nucleotide sequence of the gene SSA4 is SEQ ID NO. 8, and the nucleotide sequence of the gene SNC2 is SEQ ID NO. 9.
  5. 5. An application of the pichia pastoris gene engineering strain GGM4 in high-efficiency expression of recombinant human granulocyte-macrophage colony stimulating factor.
  6. 6. A high-density fermentation method using the pichia pastoris genetically engineered strain GGM4 of claim 1, comprising the steps of: (1) Activating Pichia pastoris gene engineering strain GGM4 in YPD culture medium, transferring to BMGY culture medium, and performing expansion culture until OD 600 reaches 6.0-8.0 to obtain seed culture solution; (2) Transferring all the seed culture solution into a 3L fermentation tank, and fermenting and culturing until the initial glycerol is exhausted; (3) Glycerol fed-batch culture, using a feed medium containing 50% w/v glycerol, at a feed rate of 6.0 mL/h,30℃ C, pH 6.0.0, aeration rate of 1.0 vvm, and rotation speed of 400-800 rpm until wet weight reaches 200-220 g/L, stopping feeding and starving for half an hour; (4) After glycerol is exhausted, methanol feed fermentation culture is started, and the culture is carried out at 29.5 ℃ and 800 rpm ℃ for 120 h, so that the induced strain expresses the recombinant human granulocyte-macrophage colony stimulating factor.
  7. 7. The fermentation process of claim 6, wherein in step (4), the methanol feed is started at a methanol feed rate of 0h, an initial 0-5 h feed rate of 2.4 mL/h, a 6-24 h feed rate of 3.6 mL/h, a 25-48 h feed rate of 4.8 mL/h, and a 49-120 h feed rate of 6.0 mL/h.
  8. 8. The fermentation method according to claim 6, wherein the dissolved oxygen content in the steps (2) to (4) is always controlled to 20% or more.

Description

Pichia pastoris gene engineering strain GGM4 and construction method and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a pichia pastoris gene engineering strain for expressing recombinant human granulocyte-macrophage colony stimulating factor and a construction method thereof. Background The colony stimulating factor (colony stimulating factor, CSF) family has three members, namely macrophage colony stimulating factor (macrophage-colony stimulating factor, M-CSF or CSF 1), granulocyte-macrophage colony stimulating factor (granulocyte macrophage-colony stimulating factor, GM-CSF or CSF 2) and granulocyte colony stimulating factor (granulocyte-colony stimulating factor, G-CSF or CSF 3), which are involved in the production of mammalian myeloid cells and are a group of glycoproteins capable of regulating hematopoietic cell proliferation and differentiation. In the pharmaceutical application, human granulocyte-macrophage colony stimulating factor CSF2 (human CSF2, hCSF for short) has important significance in host immune response defense and tumor treatment. In recent years, more and more clinical experiments show that hCSF has important effects not only in autoimmune disease treatment, bone marrow transplantation, radiation disease treatment, and the like, but also can stimulate effective antitumor response by promoting differentiation of cross-presented dendritic cells. Natural hCSF2 is a protein consisting of 127 amino acids, whose structure comprises 2 pairs of disulfide bonds, 2 predicted N-glycosylation sites and 4 predicted O-glycosylation sites, the heterogeneity of glycosylation modifications not only results in hCSF exhibiting a large difference in apparent molecular weight (about 14.5-32 kDa), but also significantly affects its physiological activity and pharmacokinetic properties. The traditional method for extracting natural hCSF2 from placenta condition culture medium is complicated in steps and low in efficiency, and severely limits the yield. Along with the successful cloning of hCSF gene, recombinant hCSF2 is prepared in large scale, which not only solves the supply problem, but also greatly promotes the physiological function research in vitro and in vivo. Recombinant hCSF (Leukine ®) expressed in saccharomyces cerevisiae is currently the only commercial recombinant hCSF2 drug approved by the united states food and drug administration (Food and Drug Administration, FDA) approved in 1991 for the treatment of neutropenia associated with stem cell transplantation and several other neutropenia caused by leukemia or its treatment. Unlike recombinant hCSF2 expressed by Saccharomyces cerevisiae, the first non-glycosylated recombinant hCSF drug (commercially known as teli) commercially available in 1997 was expressed by E.coli, and can be used for preventing and treating leukopenia caused by chemotherapy or radiotherapy, treating hematopoietic dysfunction and myelodysplastic syndrome, preventing possible infectious complications of leukopenia, and accelerating recovery of neutropenia caused by infection. In recent years, the national drug administration (National Medical Products Administration, NMPA) has successively approved a variety of recombinant hCSF drugs, such as the rienal, you Nifen, jin Lei race sources, and the like. Therefore, with the rapid increase of the market demand of the recombinant hCSF, further expansion of the productivity has become a critical issue to be solved. Disclosure of Invention The invention aims at overcoming the defects of the prior art and providing a pichia pastoris gene engineering strain GGM4 for expressing recombinant human granulocyte-macrophage colony stimulating factor, and a construction method and application thereof, so as to realize the efficient production of the recombinant human granulocyte-macrophage colony stimulating factor. In order to achieve the above purpose, the present invention provides the following technical solutions: In a first aspect, the invention provides a pichia pastoris gene engineering strain GGM4 for expressing recombinant human granulocyte-macrophage colony stimulating factor, wherein pichia pastoris GS115 strain is used as a host strain for expressing the recombinant human granulocyte-macrophage colony stimulating factor, and the nucleotide sequence of the recombinant human granulocyte-macrophage colony stimulating factor is shown as SEQ ID NO. 1. The classification of the Pichia pastoris gene engineering strain GGM4 for expressing recombinant human granulocyte-macrophage colony stimulating factor is named Pichia pastoris (syn. Komagataella phaffii), and the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 39117 and the preservation date of 2026, 01 and 04. In a second aspect, the invention provides a construction method of the pichia pastoris gene engineering strain GGM4 acco