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CN-121986110-A - Expression and purification method of human recombinant interleukin-7

CN121986110ACN 121986110 ACN121986110 ACN 121986110ACN-121986110-A

Abstract

There is provided a fusion protein characterized in that the fusion protein comprises from the 5 'end to the 3' end the elements of 5'-MBP-Furin cleavage site-rhIL-7-3', and a method for in vitro expression purification of recombinant human interleukin 7 in mammalian cells comprising the steps of a. Providing a first expression vector comprising a nucleic acid encoding said fusion protein b. Providing a second expression vector comprising a nucleic acid encoding Furin, c. Co-transfecting the first expression vector and the second expression vector into mammalian cells, fermentation culturing the cells such that the cells express recombinant human IL-7 protein d. Centrifuging to obtain a fermentation supernatant from step c as described above, e. Purifying to obtain rhIL-7 fragments by means of POROS and CHT chromatography.

Inventors

  • ZHOU JUN
  • Cao Huijin
  • Feng Zhumei
  • WU JIANSHENG

Assignees

  • 上海药明生物技术有限公司

Dates

Publication Date
20260505
Application Date
20240914
Priority Date
20230927

Claims (10)

  1. A fusion protein is characterized by comprising the following elements from the 5 'end to the 3' end, namely a 5'-MBP-Furin restriction enzyme site-rhIL-7-3'.
  2. The fusion protein of claim 1, wherein the amino acid sequence of Maltose Binding Protein (MBP) is SEQ ID NO 3, or the amino acid sequence of Furin cleavage site is SEQ ID NO 4.
  3. The fusion protein of claim 1, wherein the recombinant human IL-7 (rhIL-7) is selected from the amino acid sequence of SEQ ID NO. 4, a conservative variant thereof, or a homologous substitution.
  4. The fusion protein of claim 1, comprising the amino acid sequence of SEQ ID NO. 1.
  5. The fusion protein of claim 1, further comprising an affinity tag, preferably a His tag.
  6. The fusion protein of claim 1, further comprising a second cleavage site downstream of the Furin cleavage site and upstream of the rhIL-7 coding sequence, preferably a TEV cleavage site.
  7. A method for in vitro expression of purified recombinant human interleukin 7 in mammalian cells comprising the steps of: a. providing a first expression vector comprising a nucleic acid encoding the fusion protein of any one of claims 1-6; b. providing a second expression vector comprising a nucleic acid encoding Furin (Furin); c. co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing to enable the cells to express recombinant human IL-7 protein; d. centrifuging to obtain a fermentation supernatant from step c above; e. Purifying by POROS XS and CHT chromatography to obtain rhIL-7 fragment, and purifying by affinity tag and enzyme digestion to obtain rhIL-7.
  8. The method of claim 7, wherein the mammalian cell is a CHO-K1 cell or a HEK293 cell, or wherein the nucleic acid encoding the fusion protein is cloned into the SalI/NotI site of the first expression vector and/or the Furin protease is cloned into the SalI/NotI site of the second expression vector.
  9. The method according to claim 7, wherein the fermentation culture is transient transfection, the culture is carried out in a shaker at 150rpm for 6-7 days, the culture temperature is reduced from 36.5 ℃ to 33 ℃ 24h after the completion of the transfection, preferably the supernatant of the fermentation culture is centrifuged at a speed of 4 ℃ and 10000 revolutions (rpm), and the supernatant after centrifugation is collected.
  10. The method according to claim 7, wherein the fermentation culture is a stable transfection, the culture is carried out in a shaker at 120rpm for 14 days, the VCD reaches >10 E6/mL, the culture temperature is reduced from 36.5 ℃ to 33 ℃, preferably the supernatant of the fermentation culture is centrifuged at a speed of 4 ℃ and 10000 revolutions (rpm), and the centrifuged supernatant is collected.

Description

Expression and purification method of human recombinant interleukin-7 (1) Technical field The present invention relates to the production of recombinant human interleukin 7 (rhIL-7) in the field of immunotherapy, and in particular to a method for constructing a vector, efficiently expressing in cells, and purifying rhIL-7 from cell fermentation broth. (2) Background art IL-7 belongs to the erythropoietin family I short chain cytokines, the gene open reading frame contains 534 base pairs, totally comprises 6 exons and codes 177 amino acids, the mature IL-7 protein has 152 amino acids, the molecular weight is about 25kDa, the mature IL-7 protein contains 3N-glycosylation sites, the molecular weight of the mature IL-7 without glycosylation modification is 17.4kDa, the alpha helix is taken as the main conformational content in the molecule, the molecular core structure is adopted, and the 3-pair disulfide bond formed by 6 cysteines in the molecule is essential for the biological activity. IL-7, as a member of the immunostimulatory cytokines, plays an important role in the adaptive immune system by promoting an immune response. Such cytokines activate immune functions through the survival and differentiation of T cells and B cells, the survival of lymphocytes, and the stimulation of Natural Killer (NK) cell activity. IL-7 also regulates lymph node development by Lymphoid Tissue Induction (LTi) cells and promotes survival and division of primary T cells or memory T cells. In addition, IL-7 enhances the immune response of humans by promoting secretion of IL-2 and interferon-gamma. The receptor for IL-7 is a heterodimer and consists of IL-7Rα (CD 127) and a common gamma chain (CD 132). Gamma chains are expressed on all hematopoietic cell types, while IL-7rα is expressed primarily by lymphocytes (including B and T lymphocyte precursors, naive T cells, and memory T cells). Low expression of IL-7rα was observed on regulatory T cells compared to effector/naive T cells expressing higher levels. Thus, CD127 was used as a surface marker to distinguish between the two populations. IL-7Rα is also expressed on innate lymphocytes NK and gut-associated lymphoid tissue (GALT) -derived T cells. The IL-7Rα (CD 127) chain is shared with TSLP (tumor stroma lymphopoietin), and CD132 (gamma chain) is shared with IL-2, IL-4, IL-9, IL-15 and interleukin 21. Two major signaling pathways are induced by the Janus kinase/STAT pathway of CD127/CD132 (i.e., jak-STAT-3 and 5) and the phosphoinositide-3 kinase pathway (i.e., PI 3K-Akt). IL-7 administration is well tolerated in patients and results in expansion of CD8 and CD4 cells and a relative decrease in CD4+ T regulatory cells. Numerous animal experiments have demonstrated that IL-7 can restore immune function through steady state proliferation of peripheral cd4+ T cells and cd8+ T cells, and can promote immune reconstitution after stem cell transplantation and chemotherapy. In addition, IL-7 enhances antigen-specific T cell responses in the application of vaccines, viral infections and adoptive cell therapies. The mechanisms by which IL-7 exerts effects include down-regulation of the expression of programmed cell death factor 1 (PD-1) and cytokine signaling inhibitor protein 3 (SOCS 3) and up-regulation of the expression of the inhibitor gene BCL-2, which plays a role in acute and chronic viral infections by antagonizing the effects of the relevant inhibitor networks. The expression of IL-7 is mostly based on E.coli BL21 strain at present, and IL-7 generated by a prokaryotic expression system lacks natural sugar chain modification, has potential influence on the bioactivity of the IL-7, and limits the application of the IL-7 in clinic. Clinical trials combining the application of rhIL-7 registered worldwide today reach 15, and the application of rhIL-7 (CYT 107) now studied is produced by eukaryotic cells. However, eukaryotic cells express IL-7 with low productivity and difficult purification. Thus, there remains a great need in the art for new efficient ways of producing IL-7. (3) Summary of the invention Thus, the object of the present invention is to obtain a method for efficient production of recombinant human IL-7 protein, unlike conventional FC tag fusion. According to the invention, the water solubility and stability of the protein are improved by utilizing the MBP tag, the expression quantity of the protein is improved, and then the Furin enzyme cleavage site between MBP and IL-7 is sheared by utilizing the second cotransfection vector expression Furin enzyme, so that the IL-7 protein with a natural structure is efficiently obtained. The invention provides a fusion protein, which is characterized by comprising the following elements from a 5 'end to a 3' end, namely a 5'-MBP-Furin cleavage site-rhIL-7-3'. In a preferred embodiment of this aspect, the amino acid sequence of Maltose Binding Protein (MBP) is SEQ ID NO 3, or the amino acid sequence of the Furin cleavage site is SEQ ID