CN117069825A - Recombinant erythropoietin and application thereof - Google Patents

Recombinant erythropoietin and application thereof Download PDF

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CN117069825A
CN117069825A CN202310561356.1A CN202310561356A CN117069825A CN 117069825 A CN117069825 A CN 117069825A CN 202310561356 A CN202310561356 A CN 202310561356A CN 117069825 A CN117069825 A CN 117069825A
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mutation
amino acid
seq
recombinant erythropoietin
recombinant
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王丁力
任广彩
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Foshan Hanteng Biotechnology Co ltd
Cantonbio Co ltd
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Foshan Hanteng Biotechnology Co ltd
Cantonbio Co ltd
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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Abstract

The present invention relates to recombinant erythropoietin, nucleic acid molecules encoding same, fusion proteins comprising same, expression vectors, host cells and compositions thereof, and uses thereof. The recombinant erythropoietin of the present invention comprises mutations at positions 29, 33, 88 and/or 139 corresponding to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof. The mutated recombinant erythropoietin has improved thermal stability or in vivo binding activity, is beneficial to the industrial production thereof, and can benefit non-human mammals suffering from erythropenia to cause anemia.

Description

Recombinant erythropoietin and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to recombinant erythropoietin, nucleic acid molecules encoding the same, fusion proteins containing the same, expression vectors, host cells and compositions thereof and application thereof.
Background
Erythropoietin, also known as hematopoietin or hematopoietic element, EPO for short, is a glycoprotein hormone. It is capable of stimulating erythropoiesis, i.e., erythropoiesis. Therefore, the recombinant EPO can promote erythrocyte production, so that anemia caused by multiple factors, such as anemia caused by renal insufficiency, anemia accompanied by malignant tumor, anemia caused by rheumatism and rheumatoid, chronic anemia of serious parasitic patients, sickle cell anemia and the like can be treated.
Recombinant EPO for human use is commercially available. Commercial recombinant EPO is commercially available from Aranesp TM And Epogen TM . Current sales of human recombinant EPO are about 70-80 billion dollars per year. However, veterinary EPO for the treatment of anemia in non-human mammals remains blank. Taking dogs and cats with human lives as an example, according to incomplete statistics, over 200 ten thousand cats and 35 ten thousand dogs have chronic kidney disease, and there is insufficient EPO in the dogs or cats with chronic kidney disease to cause anemia. In anemic non-human mammals, such as dogs or cats, administration of recombinant EPO to humans to treat anemia thereof typically results in an immune response, such as in cats where anti-human is often producedProtein-like antibodies, thereby eliciting other disorders such as pure red cell aplastic anemia (PRCA) occurring in 25-30% cats. Therefore, the EPO for animals has very broad market prospect.
The EPO carried by non-human mammals is poor in thermal stability and unfavorable for production and application, so that development of the recombinant EPO with high thermal stability for animals is needed.
Disclosure of Invention
The invention aims at providing a recombinant erythropoietin with high thermal stability, a nucleic acid molecule encoding the recombinant erythropoietin, a fusion protein containing the recombinant erythropoietin, an expression vector, a host cell and a composition of the recombinant erythropoietin and application of the recombinant erythropoietin.
In a first aspect, the invention provides a recombinant erythropoietin comprising mutations at positions 29, 33, 88, and/or 139 corresponding to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof.
The term "Erythropoietin" (EPO) as used herein is also known as Erythropoietin, erythropoietin or hematopoietic hormone, an endogenous glycoprotein hormone that stimulates erythropoiesis.
The term "recombinant EPO" as used herein is consistent with the meaning of "recombinant erythropoietin".
The amino acid sequence shown in SEQ ID NO. 1 is APPRLICDSRVLERYILEAREAENVTM GC 29 A 30 QG 32 C 33 SFSENITVPDTKVNFYTWKRMDVGQQALEVWQGLALLSEAI LRGQALLANASQP 87 S 88 ETPQLHVDKAVSSLRSLTSLLRALGAQKEAMSLPEEA SPAPLRTFTVDTLC 139 KLFRIYSNFLRGKLTLYTGEACRRGDR, which is the amino acid sequence of wild-type canine EPO (wherein the superscript numbers indicate where the corresponding amino acid is located in the amino acid sequence).
The amino acid sequence shown in SEQ ID NO. 2 is APPRLICDSRVLERYILGAREAENVTM GC 29 A 30 EG 32 C 33 SFSENITVPDTKVNFYTWKRMDVGQQAVEVWQGLALLSEAI LRGQALLANSSQP 87 S 88 ETLQLHVDKAVSSLRSLTSLLRALGAQKEATSLPEATS AAPLRTFTVDTLC 139 KLFRIYSNFLRGKLTLYTGEACRRGDR, which is the amino acid sequence of wild-type cat EPO (wherein the superscript numbers indicate where the corresponding amino acid is located in the amino acid sequence).
Herein, the wild type (wild type) is relative to the mutation. In the study, individuals obtained from nature, i.e., not artificially mutagenized, were taken as wild type, and the genome carried by the wild type was the wild type genome. The EPO protein translated from the wild type carried EPO gene is the wild type EPO.
The term "ortholog" as used herein refers to the same gene or protein in different species.
In some embodiments, orthologs include: amino acid sequence of EPO in cats, macaque, pigs, cattle, sheep and horses.
In some embodiments, the ortholog is selected from erythropoietin as set forth in SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 or SEQ ID NO. 21.
In some embodiments, the recombinant erythropoietin comprises mutations at positions 29 and/or 33, and 88 and/or 139 corresponding to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof, which further increases the thermal stability of recombinant EPO.
In some embodiments, the post-mutation amino acid at position 29 is selected from S, A, G, T or P;
the 33 rd mutated amino acid is selected from S, A, G, T or P;
the 88 th mutated amino acid is C;
the 139 th mutated amino acid is selected from S, A, G or T.
In some embodiments, the mutation at position 29 is selected from C29S, C29A, C G, C29T or C29P.
In some embodiments, the mutation at position 33 is selected from C33S, C33A, C, G, C, 33T or C33P.
In some embodiments, the mutation at position 88 is S88C.
In some embodiments, the mutation at position 139 is selected from C139S, C139A, C139G or C139T.
Herein, amino acids at specific positions of an amino acid sequence are indicated by single letter abbreviations and numbers of amino acids. For example, C29S represents a mutation of C to S at site 29 of recombinant EPO. Similarly, C29A, C29G, C29T, C33S, C33A, C33G, C33T, C P, S88C, C S, C139A, C G, C T, and the following single abbreviations and numerical meanings of amino acids.
In some embodiments, the mutation is selected from any one of the following groups, wherein the groups are separated by a semicolon:
C29S; C29A; C29G; C29T; C29P; C33S; C33A; C33G; C33T; C33P; S88C; C139S; C139A; C139G; C139T; c29S, S88C; c29A, S88C; c29G, S88C; c29T, S88C; c29P, S88C; c33S, S88C; c33A, S88C; c33G, S88C; c33T, S88C; c33P, S88C; C29S, S88C, C S; C29A, S88C, C S; C29G, S88C, C S; C29T, S88C, C S; C29P, S88C, C S; c29S, S88C, C a; c29A, S88C, C a; c29G, S88C, C a; c29T, S88C, C a; c29P, S88C, C a; c29S, S88C, C G; c29A, S88C, C G; c29G, S88C, C G; c29T, S88C, C G; c29P, S88C, C G; c29S, S88C, C T; c29A, S88C, C T; c29G, S88C, C T; c29T, S88C, C T; c29P, S88C, C T; C33S, S88C, C S; C33A, S88C, C S; C33G, S88C, C S; C33T, S88C, C S; C33P, S88C, C S; C33S, S88C, C a; C33A, S88C, C a; C33G, S88C, C a; C33T, S88C, C a; C33P, S88C, C a; c33S, S88C, C G; c33A, S88C, C G; c33G, S88C, C G; c33T, S88C, C G; c33P, S88C, C G; c33S, S88C, C T; c33A, S88C, C T; c33G, S88C, C T; c33T, S88C, C T; or C33P, S88C, C T.
In some embodiments, the mutation is selected from any one of the following groups, wherein the groups are separated by a semicolon: c29S, S88C; c33P, S88C; C33P, S88C, C S; or C29S, S88C, C S.
In some embodiments, the recombinant erythropoietin further comprises at least one added and/or repositioned N-linked glycosylation modification site.
In some embodiments, the N-linked glycosylation modification site is located at the following site of the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, or an ortholog thereof:
a) Position 30; and/or
b) 88 th or 86 th.
The glycosylation level of recombinant EPO is further increased by the addition and/or repositioning of N-linked glycosylation modification sites, thereby increasing the thermal stability and in vivo half-life of recombinant EPO.
As used herein, the term "relocate" refers to a change in the N-linked glycosylation modification site of recombinant EPO relative to the N-linked glycosylation modification site of the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof.
In some embodiments, the recombinant erythropoietin further comprises mutations at positions 30, 32, and/or 87 corresponding to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof.
In some embodiments, the amino acid after mutation at position 30 is N; the 32 rd mutated amino acid is T, and the 31 st amino acid is not P. The N at position 32 of recombinant EPO is susceptible to glycosylation, thereby increasing the half-life of recombinant erythropoietin.
In some embodiments, the amino acid after mutation at position 88 is N, the amino acid at position 90 is S or T, and the amino acid at position 89 is not P. The N at position 88 of recombinant EPO is susceptible to glycosylation, thereby increasing the half-life of recombinant erythropoietin.
In some embodiments, the amino acid after mutation at position 88 is T, the amino acid at position 86 is N, and the amino acid at position 87 is not P. The N at position 86 of recombinant EPO is susceptible to glycosylation, thereby increasing the half-life of the recombinant erythropoietin.
In some embodiments, the mutation at position 30 is a30N; the 32 nd mutation is G32T.
In some embodiments, the mutation at position 87 is P87V; the 88 th mutation is S88N.
In some embodiments, the mutation at position 87 is P87V; the 86 th mutation is Q86N.
In some embodiments, the mutation further comprises a30N, G T; S88N; Q86N, P87V, S T; a30N, G32T, S88N; a30N, G32T, Q86N, P87V; or a30N, G32T, Q86N, P87V, S88T.
In some embodiments, the mutation is selected from any one of the following groups, wherein the groups are separated by a semicolon:
C29S、A30N、G32T；C29A、A30N、G32T；C29G、A30N、G32T；
C29T、A30N、G32T；C29P、A30N、G32T；C33S、A30N、G32T；C33A、A30N、G32T；C33G、A30N、G32T；C33T、A30N、G32T；C33P、A30N、G32T；A30N、G32T；C139S、A30N、G32T；C139A、A30N、G32T；C139G、A30N、G32T；C139T、A30N、G32T；C29S、P87V、S88N；C29A、P87V、S88N；C29G、P87V、S88N；C29T、P87V、S88N；C29P、P87V、S88N；
C33S、P87V、S88N；C33A、P87V、S88N；C33G、P87V、S88N；C33T、P87V、S88N；C33P、P87V、S88N；P87V、S88N；C139S、P87V、S88N；
C139A、P87V、S88N；C139G、P87V、S88N；C139T、P87V、S88N；C29S、A30N、G32T、P87V、S88N；C29A、A30N、G32T、P87V、S88N；C29G、A30N、G32T、P87V、S88N；C29T、A30N、G32T、P87V、S88N；C29P、A30N、G32T、P87V、S88N；C33S、A30N、G32T、P87V、S88N；C33A、A30N、G32T、P87V、S88N；C33G、A30N、G32T、P87V、S88N；C33T、A30N、G32T、P87V、S88N；C33P、A30N、G32T、P87V、S88N；C139S、A30N、G32T、P87V、S88N；C139A、A30N、G32T、P87V、S88N；C139G、A30N、G32T、P87V、S88N；C139T、A30N、G32T、P87V、S88N；S88C、A30N、G32T；C29S、S88C、A30N、G32T；C29A、S88C、A30N、G32T；
C29G、S88C、A30N、G32T；C29T、S88C、A30N、G32T；C29P、S88C、A30N、G32T；C33S、S88C、A30N、G32T；C33A、S88C、A30N、G32T；
c33G, S88C, A N, G T; c33T, S88C, A N, G T; c33P, S88C, A N, G T; C29S, S88C, C139S, A30N, G T; C29A, S88C, C139S, A30N, G T; C29G, S88C, C139S, A30N, G T; C29T, S88C, C139S, A30N, G T; C29P, S88C, C139S, A30N, G T; C29S, S88C, C139A, A30N, G T; C29A, S88C, C139A, A30N, G T; C29G, S88C, C139A, A30N, G T; C29T, S88C, C139A, A30N, G T; C29P, S88C, C139A, A30N, G T; C29S, S88C, C139G, A30N, G T; C29A, S88C, C139G, A30N, G T; C29G, S88C, C139G, A30N, G T; C29T, S88C, C139G, A30N, G T; C29P, S88C, C139G, A30N, G T; C29S, S88C, C139T, A30N, G T; C29A, S88C, C139T, A30N, G T; C29G, S88C, C139T, A30N, G T; C29T, S88C, C139T, A30N, G T; C29P, S88C, C139T, A30N, G T; C33S, S88C, C139S, A30N, G T; C33A, S88C, C139S, A30N, G T; C33G, S88C, C139S, A30N, G T; C33T, S88C, C139S, A30N, G T; C33P, S88C, C139S, A30N, G T; C33S, S88C, C139A, A30N, G T; C33A, S88C, C139A, A30N, G T; C33G, S88C, C139A, A30N, G T; C33T, S88C, C139A, A30N, G T; C33P, S88C, C139A, A30N, G T; C33S, S88C, C139G, A30N, G T; C33A, S88C, C139G, A30N, G T; C33G, S88C, C139G, A30N, G T; C33T, S88C, C139G, A30N, G T; C33P, S88C, C139G, A30N, G T; C33S, S88C, C139T, A30N, G T; C33A, S88C, C139T, A30N, G T; C33G, S88C, C139T, A30N, G T; C33T, S88C, C139T, A30N, G T; or C33P, S88C, C139T, A30N, G T.
In some embodiments, the mutation is selected from any one of the following groups, wherein the groups are separated by a semicolon: c29S, S88C, A N, G T; C29S, S88C, C139S, A30N, G T; C29S, S88C, C139A, A30N, G T; C29S, S88C, C139G, A30N, G T; C29S, S88C, C139T, A30N, G T; c33P, S88C, A N, G T; C33P, S88C, C139S, A30N, G T; C33P, S88C, C139A, A30N, G T; C33P, S88C, C139G, A30N, G T; or C33P, S88C, C139T, A30N, G T.
In some embodiments, the recombinant erythropoietin is a recombinant erythropoietin of a non-human mammal.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal may be a companion animal, such as a dog, cat, horse, pig, or the like.
The term "companion animal" as used herein, also referred to as pet animal, refers to any domesticated animal, including cats, dogs, horses, pigs, etc., that is owned or intended to be owned by a person at a location of some sort, particularly at home, for personal entertainment or companion purposes.
In some embodiments, the non-human mammal is selected from a canine or a feline.
In some embodiments, the recombinant erythropoietin is a polyethylene glycol modified recombinant erythropoietin.
The polyethylene glycol modification can improve the in vivo half-life of the recombinant erythropoietin and the pharmacokinetics and pharmacodynamics of the recombinant erythropoietin. Thus, recombinant EPO modified with polyethylene glycol is a long-lasting recombinant erythropoietin.
In a second aspect, the invention provides a fusion protein comprising a recombinant erythropoietin provided in the first aspect of the invention, and a fusion partner.
Compared with the recombinant erythropoietin without the fusion partner, the recombinant erythropoietin with the fusion partner prolongs the half-life period of the recombinant erythropoietin in vivo and improves the pharmacokinetics and pharmacodynamics of the recombinant erythropoietin. Thus, the fusion proteins of the invention are long-lasting recombinant erythropoietin.
In some embodiments, the fusion partner is selected from the Fc region of an immunoglobulin or albumin.
In some embodiments, the immunoglobulin is selected from IgA, igG, igM, igD or IgE.
In some embodiments, the immunoglobulin is IgG.
In some embodiments, other amino acid sequences that do not affect the activity of the fusion protein may also be added at the N-terminus or C-terminus of the fusion protein. Preferably, the other amino acid sequence does not affect the activity of the fusion protein or facilitates expression of the fusion protein (signal peptide), or facilitates purification of the fusion protein (e.g., purification tag 6×His).
In a third aspect, the invention provides a nucleic acid molecule encoding a recombinant erythropoietin provided in the first aspect of the invention or a fusion protein provided in the second aspect of the invention.
In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid molecule provided in the third aspect of the invention.
As used herein, "vector" is used to describe a polynucleotide that can be engineered to contain one or more clones that can be propagated in a host cell. The carrier may comprise one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters or enhancers) that regulate expression of the polypeptide of interest, or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in a colorimetric assay, e.g., β -galactosidase). "expression vector" refers to a vector used to express a polypeptide of interest in a host cell. The vector may be a DNA plasmid that can be delivered by a non-viral method (e.g., naked DNA, formulated DNA, or liposomes) or by a viral method.
In a fifth aspect, the invention provides a host cell comprising an expression vector provided in the fourth aspect of the invention.
As used herein, "host cell" refers to a cell that may or may not be the recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; a plant cell; insect cells. Mammalian cells include, but are not limited to, NS0 cells, HEK293 cells, and CHO cells, and derivatives thereof, such as HEK293-6E, CHODG-44, CHO-S, and CHO-K cells. Host cells include progeny of a single host cell, and due to natural, accidental, or deliberate mutation, the progeny may not necessarily be exactly identical (in morphology or in genomic DNA complement) to the original parent cell.
In a sixth aspect, the invention provides a composition comprising a recombinant erythropoietin provided in the first aspect of the invention, or a fusion protein provided in the second aspect of the invention, or a nucleic acid molecule provided in the third aspect of the invention, or an expression vector provided in the fourth aspect of the invention, or a host cell provided in the fifth aspect of the invention, and a pharmaceutically acceptable carrier.
As used herein, a "pharmaceutically acceptable carrier" may include any solvent, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic and absorption delaying agent, and the like, which are physiologically compatible. Specific examples may be one or more of water, brine, phosphate buffered saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In many cases, the pharmaceutically acceptable carrier may include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol), sodium chloride, and the like. Of course, the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, for extending the shelf life or efficacy of the antibody.
In a seventh aspect, the invention provides the use of a recombinant erythropoietin provided in the first aspect of the invention in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal.
In some embodiments, the disorder associated with reduced erythropoiesis is anemia.
In some embodiments, the anemia is anemia arising from chronic kidney disease, rheumatism or rheumatoid, parasitic infection, or malignancy, or sickle cell anemia.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal is a companion animal, such as a canine, feline, equine, porcine, or the like.
In some embodiments, the non-human mammal is a canine or a feline.
In an eighth aspect, the present invention provides the use of a fusion protein provided in the second aspect of the invention in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal.
In some embodiments, the disorder associated with reduced erythropoiesis is anemia.
In some embodiments, the anemia is anemia arising from chronic kidney disease, rheumatism or rheumatoid, parasitic infection, or malignancy, or sickle cell anemia.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal is a companion animal, such as a canine, feline, equine, porcine, or the like.
In some embodiments, the non-human mammal is a canine or a feline.
In a ninth aspect, the invention provides the use of a nucleic acid molecule provided in the third aspect of the invention in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal.
In some embodiments, the disorder associated with reduced erythropoiesis is anemia.
In some embodiments, the anemia is anemia arising from chronic kidney disease, rheumatism or rheumatoid, parasitic infection, or malignancy, or sickle cell anemia.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal is a companion animal, such as a canine, feline, equine, porcine, or the like.
In some embodiments, the non-human mammal is a canine or a feline.
In a tenth aspect, the present invention provides the use of an expression vector as provided in the fourth aspect of the invention in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal.
In some embodiments, the disorder associated with reduced erythropoiesis is anemia.
In some embodiments, the anemia is anemia arising from chronic kidney disease, rheumatism or rheumatoid, parasitic infection, or malignancy, or sickle cell anemia.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal is a companion animal, such as a canine, feline, equine, porcine, or the like.
In some embodiments, the non-human mammal is a canine or a feline.
In an eleventh aspect, the invention provides the use of a host cell provided in the fifth aspect of the invention in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal.
In some embodiments, the disorder associated with reduced erythropoiesis is anemia.
In some embodiments, the anemia is anemia arising from chronic kidney disease, rheumatism or rheumatoid, parasitic infection, or malignancy, or sickle cell anemia.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal is a companion animal, such as a canine, feline, equine, porcine, or the like.
In some embodiments, the non-human mammal is a canine or a feline.
In a twelfth aspect, the invention provides the use of a composition provided in the sixth aspect of the invention in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal.
In some embodiments, the disorder associated with reduced erythropoiesis is anemia.
In some embodiments, the anemia is anemia arising from chronic kidney disease, rheumatism or rheumatoid, parasitic infection, or malignancy, or sickle cell anemia.
In some embodiments, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
In some embodiments, the non-human mammal is a companion animal, such as a canine, feline, equine, porcine, or the like.
In some embodiments, the non-human mammal is a canine or a feline.
The recombinant erythropoietin provided by the invention comprises mutations at positions 29, 33, 88 and/or 139 corresponding to the amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or ortholog thereof, so that the thermal stability of the erythropoietin is improved, the EPO industrial production is facilitated, and the non-human mammal suffering from anemia caused by erythropenia benefits.
Drawings
FIG. 1 shows the results of non-reducing SDS-PAGE of the culture supernatants of the fed batches.
FIG. 2 is a Ni column purification scheme of recombinant canine EPO protein molecule IV008-09. The arrows in the figure represent IV008-09.
FIG. 3 shows the SEC purification results of wild type canine EPO protein molecule IV 008-01.
FIG. 4 shows the SEC purification results of recombinant canine EPO protein molecule IV 008-08.
FIG. 5 shows the SEC purification results of recombinant canine EPO protein molecule IV008-09.
FIG. 6 shows the SEC purification results of recombinant canine EPO protein molecule IV 008-10.
FIG. 7 shows the SEC purification results of recombinant canine EPO protein molecule IV 008-11.
FIG. 8 shows the results of non-reducing SDS-PAGE of purified samples.
FIG. 9 shows the detection result of the fluorescence thermal drift Tm value of the purified sample.
FIG. 10 shows the Tagg value detection result of purified sample IV 008-01.
FIG. 11 shows the Tagg value detection results of purified sample IV 008-08.
FIG. 12 shows the Tagg value measurement results of purified sample IV 008-10.
FIG. 13 shows the Tagg value measurement results of purified sample IV 008-11.
Detailed Description
The invention is further illustrated by the following examples, it being understood that the examples of the invention are presented by way of illustration only and not by way of limitation, and that simple modifications of the invention are within the scope of the invention as claimed.
The gist of the present invention is the finding that the obtained recombinant erythropoietin is capable of increasing the thermal stability of erythropoietin corresponding to the mutations at positions 29, 33, 88 and/or 139 of the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof, in order to facilitate the treatment of diseases associated with reduced erythropoiesis in non-human mammals, such as anemia.
The recombinant erythropoietin has a higher thermal stability when it comprises mutations at positions 29 and/or 33 and 88 and/or 139 corresponding to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof.
On this basis, glycosylation of at least one amino acid of recombinant erythropoietin can further improve the thermal stability of recombinant erythropoietin.
The mutation site corresponds to the amino acid sequence shown as SEQ ID NO. 1, namely wild type canine EPO, the amino acid sequence shown as SEQ ID NO. 2, namely wild type feline EPO, but is not limited to wild type feline or canine EPO, and the amino acids at positions 29, 33, 88 and/or 139 of the ortholog of wild type feline or canine EPO are also within the scope of the present invention as set forth in the technical scheme. This can be readily determined by one skilled in the art through sequence alignment.
In order to illustrate that the recombinant erythropoietin provided by the invention is suitable for not only the amino acid sequences of canine and feline EPO, but also the amino acid sequences of other non-human mammals, the invention further provides the amino acid sequences of EPO of other non-human mammals as follows:
the amino acid sequence of EPO of wild macaque is: APPRLICDSRVLERYILEAREAENVTM GC 29 A 30 QG 32 C 33 SFSENITVPDTKVNFYTWKRMDVGQQALEVWQGLALLSEAI LRGQALLANASQPSETPQLHVDKAVSSLRSLTSLLRALGAQKEAMSLPEEASP APLRTFTVDTLC 139 KLFRIYSNFLRGKLTLYTGEACRRGDR(SEQ ID NO:17)。
The amino acid sequence of wild type porcine EPO is: APPRLICDSRVLERYILEAKEGENATMGC 29 A 30 ESC 33 SFSENITVPDTKVNFYAWKRMEVQQQAMEVWQGLALLSEAILQG QALLANSSQP 87 S 88 EALQLHVDKAVSGLRSLTSLLRALGAQKEAIPLPDASPSSA TPLRTFAVDTLC 139 KLFRNYSNFLRGKLTLYTGEACRRRDR(SEQ ID NO:18)。
The amino acid sequence of wild bovine EPO is: APARLICDSRVLERYILEAREAENATMGC 29 A 30 EG 32 C 33 SFNENITVPDTKVNFYAWKRMEVQQQALEVWQGLALLSEAILR GQALLANASQP 87 CEALRLHVDKAVSGLRSLTSLLRALGAQKEAISLPDATPSA APLRAFTVDALSKLFRIYSNFLRGKLTLYTGEACRRGDR(SEQ ID NO:19)。
The amino acid sequence of EPO of the wild sheep is as follows: APPRLICDSRVLERYILEAREAENATMGC 29 A 30 EG 32 C 33 SFSENITVPDTKVNFYAWKRMEVQQQALEVWQGLALLSEAIFRG QALLANASQP 87 C 88 EALRLHVDKAVSGLRSLTSLLRALGAQKEAIPLPDATPSA APLRIFTVDALSKLFRIYSNFLRGKLTLYTGEACRRGDR (SEQ ID NO: 20). The wild type equine EPO amino acid sequence is: APPRLICDSRVLERYILEAREAENVTMGC 29 A 30 EG 32 C 33 SFGENVTVPDTKVNFYSWKRMEVEQQAVEVWQGLALLSEAILQGQ ALLANSSQP 87 S 88 ETLRLHVDKAVSSLRSLTSLLRALGAQKEAISPPDAASAAPL RTFAVDTLC 139 KLFRIYSNFLRGKLKLYTGEACRRGDR(SEQ ID NO:21)。
The following examples relate to the amino acid sequence of EPO from wild type dogs as shown in SEQ ID NO. 1 and the amino acid sequence of EPO from wild type cats as shown in SEQ ID NO. 2.
TABLE 1 conservative amino acid substitutions
Amino acid residues Conservative substitutions Preferably conservative substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；Nle Leu
Leu(L) Nle；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；Nle Leu
The recombinant EPO of the present invention or orthologs thereof may also be substituted with conservative amino acids as shown in Table 1 above without affecting the activity or effect thereof.
EXAMPLE 1 construction of recombinant vector encoding recombinant EPO
The polynucleotide of recombinant canine or feline EPO is synthesized on a pUC57 vector, the recombinant plasmid is digested by EcoRI/HindIII, and the recombinant canine or feline EPO gene fragment is connected on a eukaryotic expression vector pEE12.4, and the recombinant canine or feline EPO gene fragment is used for transfection of CHO-K1 (Merck) cells after the sequencing is correct.
The procedure for constructing the recombinant vector containing the polynucleotide of wild type canine or feline EPO and the recombinant vector of the polynucleotide of each recombinant canine or feline EPO in example 1 was the same, except that the EPO used was different. The polynucleotide encoding recombinant EPO described below was constructed into vector pEE12.4 to obtain a recombinant vector expressing the polynucleotide of recombinant EPO.
Wild-type canine EP O (IV 008-01) sequence containing signal peptide (italic and underlined) and tag (underlined only) is shown in SEQ ID NO: 3:
mutant canine EP O-C33P, S88C (IV 008-02) containing a signal peptide (shown in italics and underlined) and a tag (shown only in underlined) has the sequence shown in SEQ ID NO: 4:
the sequence of the mutant canine EP O-C29S, S88C (IV 008-03) containing a signal peptide (shown in italics and underlined) and a tag (shown in underlined only) is shown in SEQ ID NO: 5:
the sequences of mutant dogs EP O-C33P, S88C, C139S (IV 008-04) containing a signal peptide (shown in italics and underlined) and a tag (shown in underlined only) are shown in SEQ ID NO: 6:
Mutant canine EP O-C29S, S88C, C139S (IV 008-05) sequences containing signal peptide (italics and underlined) and tag (underlined only) are shown in SEQ ID NO: 7:
containing signal peptides (italics and underlined)Shown) and tags (shown underlined only), mutant canine EP O- (A30N, G32T, P87V, S88N)&The sequence of C33P (IV 008-08) is shown as SEQ ID NO. 8:
mutant canine EP O- (A30N, G32T, P87V, S88N) containing a signal peptide (shown in italics and underlined) and a tag (shown only underlined)&The sequence of C29S (IV 008-09) is shown as SEQ ID NO: 9:
mutant canine EP O- (A30N, G32T) containing a signal peptide (shown in italics and underlined) and a tag (shown only in underlined)&The sequence of C33S, S88C and C139S (IV 008-10) is shown in SEQ ID NO: 10:
mutant canine EP O- (A30N, G32T) containing a signal peptide (shown in italics and underlined) and a tag (shown only in underlined)&The sequence of C29S, S88C and C139S (IV 008-11) is shown in SEQ ID NO: 11:/>
wild-type cat EP O sequence SEQ ID NO. 12 containing a signal peptide (italic and underlined) and a tag (underlined only) is as follows:
mutant cat EPO-C33P, S88C sequence containing signal peptide (italic and underlined) and tag (underlined only) is shown as SEQ ID NO: 13:
Mutant cat EPO-C29S, S88C sequences containing signal peptide (italic and underlined) and tag (underlined only) are shown in SEQ ID NO: 14:
mutant cat EPO-C33P, S88C, C139S sequences containing signal peptide (italic and underlined) and tag (underlined only) are shown in SEQ ID NO: 15:
mutant cat EPO-C29S, S88C, C139S sequences SEQ ID NO 16 containing signal peptide (italics and underlined) and tag (underlined only) are as follows:/>
EXAMPLE 2 establishment of recombinant canine EPO-expressing cell lines
Chinese hamster ovary cells CHO-K1 (Merck) were used as host cells for recombinant canine EPO expression. The recombinant vector was transfected into CHO cells. The cell pool was further screened under increasing pressure. And selecting a cell pool with the highest recombinant canine EPO protein expression for expressing recombinant canine EPO, and carrying out fed-batch culture to express the recombinant canine EPO. The results of non-reducing SDS-PAGE of the culture supernatants of the fed batch are shown in FIG. 1.
EXAMPLE 3 establishment of purification of recombinant canine EPO protein
The recombinant canine EPO protein molecules contained in the supernatant were first isolated by Ni column, and the supernatant was collected from serum-free medium in which CHO cells expressing recombinant canine EPO were cultured. The isolated protein was further purified by gel filtration in Superdex 75.
The Ni column purification patterns of 5 molecules are not obviously different, and only one of the IV008-011 Ni column purification patterns is shown here, and the detail is shown in figure 2. From the results shown in FIG. 2, it can be seen that purified recombinant canine EPO protein molecules were obtained.
Cell pool yield was calculated from the one-step purification results, which are shown in table 2:
TABLE 2
Molecular numbering Cell pool yield (g/L)
IV008-01 1.1
IV008-08 0.9
IV008-09 0.6
IV008-10 1.1
IV008-11 1.1
The SEC purification results are shown in FIGS. 3-7, and the SEC purity of mutants IV008-08 and IV008-10 and the SEC purity of IV008-11 are close to that of wild type IV008-01, and the peak time is close (wherein the abscissa starting time of FIGS. 4-7 is not 0, and the peak time is calculated by taking injection as the starting time), so that the apparent molecular weight of the protein is not obviously influenced by the mutation.
The non-reducing SDS-PAGE results of the purified samples are shown in FIG. 8, and the bands of the mutants IV008-08, IV008-10 and IV008-11 are close to the bands of the wild type IV008-01, and no obvious protein polymer is formed, which indicates that mismatch disulfide bond is not formed.
The melting temperature (Tm) of a protein is a measure of the structural heat stability of an antibody, and the higher the Tm, i.e. the higher the temperature required for denaturation of the protein, the better the heat stability of the protein. The higher the protein initiation aggregation temperature (Tagg value), i.e., the higher the temperature at which the protein begins to aggregate, the higher the colloidal stability of the antibody preparation. Both Tm and Tagg values are differences in antibody stability upon rapid exposure to heat stimulation. In this example, the Tm value of recombinant EPO was determined by fluorescence thermal drift, and the Tagg value of recombinant EPO was determined by DLS detection.
The fluorescent Thermal drift detection is to transfer the nucleic acid melting curve experimental principle of qPCR into protein research, so that the research on the Thermal stability of the protein can be conveniently carried out on a fluorescent quantitative PCR instrument through a Thermal drift experiment of the protein, and only the nucleic acid fluorescent dye is required to be replaced by SYPRO Orange commonly used for protein labeling. The SYPRO Orange fluorescent dye is a naturally quenched dye, and can be efficiently combined with an exposed protein hydrophobic core after protein denaturation, so that the fluorescent signal is enhanced.
The detection results of the fluorescence thermal drift Tm value of the purified sample are shown in FIG. 9 and Table 3.
TABLE 3 Table 3
Molecular numbering Tm(℃)
PBS(Buffer) N/A
IV008-01 55.3
IV008-08 48
IV008-10 62.9
IV008-11 70.3
The results of the detection of Tagg values of the purified samples are shown in fig. 10 to 13.
According to the detection result of the Tm value and the detection result of the Tagg value, the Tagg value has a good corresponding relation with the Tm value, meanwhile, IV008-01 and IV008-08 generate serious aggregation in the heating process, the diameter of protein particles can reach hundreds of nanometers, IV008-10 and IV008-11 only have tens of nanometers, and IV008-11 is more below 20 nm. It can be seen that the thermal stability of IV008-01, IV008-08, IV008-10 and IV008-11 are progressively higher.
In summary, the expression levels of IV008-08 and IV008-09 were slightly decreased and the thermal stability of IV008-08 was slightly decreased as compared with IV008-01 (wild type canine EPO); the expression levels of IV008-10 and IV008-11 are basically unchanged, but the thermal stability is improved obviously, and it is speculated that on the basis of the amino acid sequence shown in SEQ ID NO. 1, the 88 th S is mutated into C, so that the introduction of disulfide bonds obviously improves the thermal stability of EPO, wherein when the 33 th amino acid is C and the 88 th amino acid is C, the thermal stability of EPO is higher, the Tm value is improved by 15 ℃, and the Tagg value is improved by 16.33 ℃.
The experimental results of the above examples show that the provided recombinant EPO has high thermal stability, and is beneficial to the production of recombinant EPO for animals, so that the recombinant EPO benefits from non-human mammals, such as companion animals, dogs, cats, horses, etc., suffering from diseases associated with reduced erythropoiesis, such as anemia. Wherein, the Tm value and the Tagg value of IV008-10 and IV008-11 are obviously increased relative to wild type canine EPO (IV 008-01). The Tm values of IV008-10 and IV008-11 are raised by 7.6℃and 15℃respectively, relative to the Tm value of IV 008-01. The Tagg values of IV008-10 and IV008-11 increased by 6.98℃and 16.33℃respectively, relative to the Tagg value of IV 008-01.
EXAMPLE 4 in vivo and in vitro Activity detection of recombinant EPO protein
The purity of the purified recombinant canine EPO protein samples is shown in table 4:
TABLE 4 Table 4
Molecular numbering Concentration (mg/mL)
IV008-01 3.36
IV008-08 4.75
IV008-10 4.56
IV008-11 4.75
(1) Recombinant canine EPO protein in vitro activity detection
This example uses a canine Erythropoietin (EPO) ELISA kit to detect in vitro activity of a sample.
A. Material preparation
1) Equilibrate the kit to room temperature;
2) Diluting the concentrated wash solution (1:25) with double distilled water;
3) Preparing a standard substance: adding 1.0mL of standard diluent to the freeze-dried standard, standing for 10min, and lightly mixing after the diluent is fully dissolved, wherein the tube is marked as (1);
4) Standard substance dilution: taking 7 clean reagent tubes, respectively labeling (2) (3) (4) (5) (6) (7) (8), and adding 300 mu L of standard substance diluent into each tube; taking 300 mu L from the tube (1) to the tube (2), mixing uniformly, taking 300 mu L from the tube (2), adding into the tube (3), and analogizing to the tube (7); (8) the tube was a standard dilution used as a negative control;
5) Preparing a biotinylated antibody working solution: diluting and concentrating the biotinylated antibody (1:100) by using an antibody diluent to prepare a biotinylated antibody working solution;
6) Preparing an enzyme conjugate working solution: diluting the concentrated enzyme conjugate (1:100) with an enzyme conjugate diluent to prepare an enzyme conjugate working solution;
7) Preparing TMB color development working solution: TMB color developing solution A and TMB color developing solution B are prepared according to the following steps of 9:1, mixing and preparing TMB color development working solution.
B. Sample washing
Washing the automatic plate washing machine: injecting 350 mu L of washing liquid into each hole, wherein the interval between injection and suction is 20-30 seconds;
and (3) manually washing: each well was filled with 350. Mu.l of the washing solution, left for 30 seconds, and the liquid in the well was discarded, and the absorbent paper was blotted dry.
C. In vitro Activity assay
1) Adding a pre-prepared sample and a standard substance, and reacting for 90min at 37 ℃;
2) Washing the sample for 2 times, adding a biotinylated antibody working solution, and reacting for 60min at 37 ℃;
3) Washing the sample for 3 times, adding ABC working solution, and reacting for 30min at 37 ℃;
4) Washing the sample for 5 times, adding TMB color developing solution, and reacting at 37 ℃;
5) Adding TMB stopping solution;
6) And (5) measuring an OD value by an enzyme label instrument within 10min, and calculating the concentration and activity of the sample.
The in vitro activity detection results of the recombinant canine EPO protein are shown in Table 5:
TABLE 5
From the results of the above binding activity assays, it was found that the wild-type canine EPO protein has comparable in vitro binding activity to the recombinant canine EPO protein, which shows that the mutation that results in improved thermostability of the recombinant canine EPO protein does not affect its in vitro binding activity.
(2) Recombinant canine EPO protein in vivo activity assay
This example evaluates recombinant canine EPO protein in vivo activity by measuring reticulocyte fraction (RET%) (i.e., the ratio of reticulocytes to total number of erythrocytes) in mice. Reticulocytes are precursors of erythrocytes, which belong to erythrocytes that have not yet fully matured. The ratio of reticulocytes to total number of erythrocytes reflects the function of erythropoiesis and thus can evaluate the in vivo activity of recombinant canine EPO protein.
A. Material preparation
Dilution liquid: weighing 0.1g of bovine serum albumin, adding 0.85-0.90% sodium chloride solution for dissolution and dilution to 100mL to obtain the required diluent.
Anticoagulant: weighing 100mg of ethylene diamine tetraacetic acid dipotassium, adding 10mL of 0.85% -0.90% sodium chloride solution for dissolution, and uniformly mixing to obtain the required anticoagulant.
B. In vivo Activity detection
Pre-experiment: diluting the sample to an initial concentration of 0.5-0.6 mg/mL by using a diluent, performing gradient dilution of 10 times (0.05-0.06), 20 times (0.025-0.03) and 40 times (0.0125-0.015), and taking blood after injecting the sample into a mouse body for 4 days, wherein the result shows that the reticulocyte ratio of the sample is the same level as that of human erythropoietin;
sample dilution:
the initial concentrations of each batch of samples (sample parameters are shown in Table 6) were diluted to 0.5mg/mL and subjected to 25-fold, 50-fold and 100-fold gradient dilutions.
TABLE 6
Molecular numbering Concentration (mg/mL) Initial concentration (mg/mL) Dilution factor
IV008-01 3.36 0.5 6.7
IV008-08 4.75 0.5 9.5
IV008-10 4.56 0.5 9.1
IV008-11 4.75 0.5 9.5
The experimental set after sample dilution was set as in Table 7, and the diluted sample was injected into mice and collected from the orbits of the mice after 4 daysDrop-wise, place in a blood collection tube to which 200. Mu.L of ethylenediamine tetraacetic acid dipotassium anticoagulant was previously added. Anticoagulation was taken and the results of the Ret in the blood of each mouse were counted using a fully automatic reticulocyte analyzer and are shown in Table 8.
The specific activity of the samples calculated by parallel line measurement of the dose (IU) to Ret% (rule 1431 in the edition 2020 of Chinese pharmacopoeia) is shown in Table 9.
TABLE 7
Sample group Number of mice Injection quantity Dosage of
100 times sample 8 pieces/group 0.5mL of only 2.5 μg/mouse
50 times sample 8 pieces/group 0.5 mL/min 5 μg/mouse
25 times sample 8 pieces/group 0.5 mL/min 10 μg/mouse
TABLE 8
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TABLE 9
Molecular numbering Detecting items Detection method Specific activity (IU/mg)
IV008-01 Biological Activity in vivo Reticulocyte method 4244.6
IV008-08 Biological Activity in vivo Reticulocyte method 6826.1
IV008-10 In vivo productionPhysical activity Reticulocyte method 4026.7
IV008-11 Biological Activity in vivo Reticulocyte method 4760.1
According to the results of Table 9, the in vivo binding activity of the recombinant canine EPO protein was higher than that of the wild-type canine EPO protein except that IV008-10 was slightly lower than that of the wild-type, especially IV008-08, indicating that the mutation sites and patterns as shown in the present disclosure do not result in inactivation of the corresponding protein.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims (17)

1. A recombinant erythropoietin characterized in that said recombinant erythropoietin comprises a mutation at positions 29, 33, 88, and/or 139 corresponding to the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or an ortholog thereof.
2. The recombinant erythropoietin according to claim 1, characterized in that it comprises mutations at positions 29 and/or 33 and at positions 88 and/or 139 corresponding to the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or an ortholog thereof.
3. The recombinant erythropoietin of claim 1, wherein,
the amino acid after 29 th mutation is selected from S, A, G, T or P; and/or
The 33 rd mutated amino acid is selected from S, A, G, T or P; and/or
The 88 th mutated amino acid is C; and/or
The 139 th mutated amino acid is selected from S, A, G or T;
preferably, the mutation at position 29 is selected from C29S, C A, C29G, C T or C29P;
Preferably, the mutation at position 33 is selected from C33S, C A, C33G, C T or C33P;
preferably, the mutation at position 88 is S88C;
preferably, the mutation at position 139 is selected from C139S, C139A, C139G or C139T.
4. The recombinant erythropoietin according to claim 3, wherein the mutation is selected from any one of the group consisting of:
C29S; or C29A; or C29G; or C29T; or C29P; or C33S; or C33A; or C33G; or C33T; or C33P; or S88C; or C139S; or C139A; or C139G; or C139T; or C29S, S88C; or C29A, S88C; or C29G, S88C; or C29T, S88C; or C29P, S88C; or C33S, S88C; or C33A, S88C; or C33G, S88C; or C33T, S88C; or C33P, S88C; or C29S, S88C, C S; or C29A, S88C, C S; or C29G, S88C, C S; or C29T, S88C, C S; or C29P, S88C, C S; or C29S, S88C, C a; or C29A, S88C, C a; or C29G, S88C, C a; or C29T, S88C, C a; or C29P, S88C, C a; or C29S, S88C, C G; or C29A, S88C, C G; or C29G, S88C, C G; or C29T, S88C, C G; or C29P, S88C, C G; or C29S, S88C, C T; or C29A, S88C, C T; or C29G, S88C, C T; or C29T, S88C, C T; or C29P, S88C, C T; or C33S, S88C, C S; or C33A, S88C, C S; or C33G, S88C, C S; or C33T, S88C, C S; or C33P, S88C, C S; or C33S, S88C, C a; or C33A, S88C, C a; or C33G, S88C, C a; or C33T, S88C, C a; or C33P, S88C, C a; or C33S, S88C, C G; or C33A, S88C, C G; or C33G, S88C, C G; or C33T, S88C, C G; or C33P, S88C, C G; or C33S, S88C, C T; or C33A, S88C, C T; or C33G, S88C, C T; or C33T, S88C, C T; or C33P, S88C, C T;
Preferably, the mutation is selected from any one of the following groups:
C29S、S88C；
C33P、S88C；
C33P, S88C, C S; or (b)
C29S、S88C、C139S。
5. The recombinant erythropoietin according to claim 1, further comprising at least one added and/or relocated N-linked glycosylation modification site;
preferably, the N-linked glycosylation modification site is located at the following site of the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or an ortholog thereof:
a) Position 30; and/or
b) 88 th or 86 th.
6. The recombinant erythropoietin according to claim 5, further comprising a mutation at position 30, 32, and/or 87 corresponding to the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or an ortholog thereof;
preferably, the amino acid after mutation at position 30 is N; the 32 rd mutated amino acid is T, and the 31 st amino acid is not P;
preferably, the 88 th mutated amino acid is N, the 90 th amino acid is S or T, and the 89 th amino acid is not P;
preferably, the amino acid after 88 th mutation is T, the 86 th amino acid is N, and the 87 th amino acid is not P;
Preferably, the mutation at position 30 is a30N; the 32 nd mutation is G32T;
preferably, the mutation at position 87 is P87V; the 88 th mutation is S88N;
preferably, the mutation at position 87 is P87V; the 86 th mutation is Q86N;
preferably, the mutation further comprises any one of the following groups:
A30N、G32T；
S88N；
Q86N、P87V、S88T；
A30N、G32T、S88N；
a30N, G32T, Q86N, P87V; or (b)
A30N、G32T、Q86N、P87V、S88T。
7. The recombinant erythropoietin according to claim 6, wherein the mutation is selected from any one of the group consisting of:
c29S, A30N, G T; or C29A, A30N, G T; or C29G, A30N, G T; or C29T, A30N, G T; or C29P, A30N, G T; or C33S, A30N, G T; or C33A, A30N, G T; or C33G, A30N, G T; or C33T, A30N, G T; or C33P, A30N, G T; or a30N, G T; or C139S, A30N, G T; or C139A, A30N, G T; or C139G, A30N, G T; or C139T, A30N, G T; or C29S, P87V, S88N; or C29A, P87V, S88N; or C29G, P87V, S88N; or C29T, P87V, S88N; or C29P, P87V, S88N; or C33S, P87V, S88N; or C33A, P87V, S88N; or C33G, P87V, S88N; or C33T, P87V, S88N; or C33P, P87V, S88N; or P87V, S88N; or C139S, P87V, S88N; or C139A, P87V, S88N; or C139G, P87V, S88N; or C139T, P87V, S88N; or C29S, A30N, G T, P87V, S88N; or C29A, A30N, G T, P87V, S88N; or C29G, A30N, G T, P87V, S88N; or C29T, A30N, G T, P87V, S88N; or C29P, A30N, G T, P87V, S88N; or C33S, A30N, G T, P87V, S88N; or C33A, A30N, G T, P87V, S88N; or C33G, A30N, G T, P87V, S88N; or C33T, A30N, G T, P87V, S88N; or C33P, A30N, G T, P87V, S88N; or C139S, A30N, G T, P87V, S N; or C139A, A30N, G T, P87V, S N; or C139G, A30N, G T, P87V, S N; or C139T, A30N, G T, P87V, S N; or S88C, A30N, G T; or C29S, S88C, A N, G T; or C29A, S88C, A N, G T; or C29G, S88C, A N, G T; or C29T, S88C, A N, G T; or C29P, S88C, A N, G T; or C33S, S88C, A30N, G T; or C33A, S88C, A30N, G T; or C33G, S88C, A30N, G T; or C33T, S88C, A30N, G T; or C33P, S88C, A30N, G T; or C29S, S88C, C139S, A30 6232T; or C29A, S88C, C139S, A30 6232T; or C29G, S88C, C139S, A30 6232T; or C29T, S88C, C139S, A30 6232T; or C29P, S88C, C139S, A30 6232T; or C29S, S88C, C139A, A30 6232T; or C29A, S88C, C139A, A30 6232T; or C29G, S88C, C139A, A30 6232T; or C29T, S88C, C139A, A30 6232T; or C29P, S88C, C139A, A30 6232T; or C29S, S88C, C139G, A30 6232T; or C29A, S88C, C139G, A30 6232T; or C29G, S88C, C139G, A30 6232T; or C29T, S88C, C139G, A30 6232T; or C29P, S88C, C139G, A30 6232T; or C29S, S88C, C139T, A30 6232T; or C29A, S88C, C139T, A30 6232T; or C29G, S88C, C139T, A30 6232T; or C29T, S88C, C139T, A30 6232T; or C29P, S88C, C139T, A30 6232T; or C33S, S88C, C139S, A30N, G T; or C33A, S88C, C139S, A30N, G T; or C33G, S88C, C139S, A30N, G T; or C33T, S88C, C139S, A30N, G T; or C33P, S88C, C139S, A30N, G T; or C33S, S88C, C139A, A30N, G T; or C33A, S88C, C139A, A30N, G T; or C33G, S88C, C139A, A30N, G T; or C33T, S88C, C139A, A30N, G T; or C33P, S88C, C139A, A30N, G T; or C33S, S88C, C139G, A30N, G T; or C33A, S88C, C139G, A30N, G T; or C33G, S88C, C139G, A30N, G T; or C33T, S88C, C139G, A30N, G T; or C33P, S88C, C139G, A30N, G T; or C33S, S88C, C139T, A30N, G T; or C33A, S88C, C139T, A30N, G T; or C33G, S88C, C139T, A30N, G T; or C33T, S88C, C139T, A30N, G T; or C33P, S88C, C139T, A30N, G T;
Preferably, the mutation is selected from any one of the following groups:
c29S, S88C, A N, G T; or C29S, S88C, C139S, A30 6232T; or C29S, S88C, C139A, A30 6232T; or C29S, S88C, C139G, A30 6232T; or C29S, S88C, C139T, A30 6232T; or C33P, S88C, A30N, G T; or C33P, S88C, C139S, A30N, G T; or C33P, S88C, C139A, A30N, G T; or C33P, S88C, C139G, A30N, G T; or C33P, S88C, C139T, A30N, G T.
8. The recombinant erythropoietin according to claim 1, wherein said recombinant erythropoietin is a recombinant erythropoietin of a non-human mammal;
preferably, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
9. The recombinant erythropoietin according to claim 8, wherein said ortholog is selected from the group consisting of erythropoietin as shown in SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 or SEQ ID NO. 21.
10. The recombinant erythropoietin according to any one of claims 1-9, wherein the recombinant erythropoietin is a polyethylene glycol modified recombinant erythropoietin.
11. A fusion protein comprising the recombinant erythropoietin of any one of claims 1-9, and a fusion partner.
12. The fusion protein of claim 11, wherein the fusion partner is selected from the group consisting of an Fc region of an immunoglobulin or an albumin;
preferably, the immunoglobulin is selected from IgA, igG, igM, igD or IgE;
more preferably, the immunoglobulin is IgG.
13. A nucleic acid molecule encoding the recombinant erythropoietin of any one of claims 1-9 or the fusion protein of claim 11 or 12.
14. An expression vector comprising the nucleic acid molecule of claim 13.
15. A host cell comprising the expression vector of claim 14.
16. A composition comprising the recombinant erythropoietin of any one of claims 1-10, or the fusion protein of claim 11 or 12, or the nucleic acid molecule of claim 13, or the expression vector of claim 14, or the host cell of claim 15, and a pharmaceutically acceptable carrier.
17. Use of a recombinant erythropoietin according to any one of claims 1-10, a fusion protein according to claim 11 or 12, a nucleic acid molecule according to claim 13, an expression vector according to claim 14, a host cell according to claim 15, or a composition according to claim 16 in the manufacture of a medicament for the treatment of a disease associated with reduced erythropoiesis in a non-human mammal;
preferably, the non-human mammal is selected from the group consisting of canine, feline, cynomolgus, porcine, bovine, ovine, and equine.
CN202310561356.1A 2022-05-17 2023-05-17 Recombinant erythropoietin and application thereof Pending CN117069825A (en)

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