Recombinant human dyad stem cell factor and preparing thereof

Abstract

A recombinant human diad stem cell factor (rhdSCF) used for radiotherapy, chemicotherapy and recreating and restoring the hematognesis and immune function after bone marrow transplantation is disclosed. It sequentially contains amino acid codons No1-165 of human SCF, 12 amino acid linker peptides, codons No1-145 of human SCF and DNA fragment of termination codon. Its advantage is high specific activity.

Description

1. Technical field

[0001] The invention belongs to the technical field of polypeptide medicine prepared by genetic engineering. 2. Background technology

[0002] Stem cell factor (SCF, also called Kit ligand, Steel factor, mast cell growth factor, etc.) is a multifunctional cytokine that can cooperate with other cytokines at the multi-level hematopoietic level to promote hematopoietic stem / progenitor cells and various blood cells. It can also maintain the survival and proliferation of melanocytes, blast cells and mast cells, and promote their proliferation and differentiation. Its target cells include pluripotent hematopoietic stem / progenitor cells, erythroid cells, megakaryocytes, monocyte-macrophages, some lymphoid precursor cells, mast cells, melanocytes and blast cells.

[0003] Natural SCF is a kind of glycoprotein, which is mainly expressed by stromal cells, fibroblasts and endothelial cells, and the ratio of membrane-bound SCF and soluble SCF expression varies greatl...

Examples

specific Embodiment approach 2

[0030] Steps 1 and 2 are the same as steps 1 and 2 in Embodiment 1

[0031] 3. With a doubling time of 19 hours, living cells account for 95%, and a density of 1.5×10 6 Cells / mL 40 mL of Sf9 suspension cells cultured in SF900II medium containing 4% newborn bovine serum were centrifuged at 500 rpm for 10 minutes, and the collected cells were resuspended in 40 mL of SF900II serum-free medium. According to the multiplicity of infection (MOI) = 0.1, the titer was 1.0×10 7 0.6 ml of the recombinant virus AcNPV-rhdSCF stock solution per ml. The rotating speed of the stirring blade was 60 rpm, and after culturing at 27° C. for 48 hours, centrifuge at 10,000 rpm for 5 minutes, discard the cells and collect 40 ml of the supernatant. The activity of the fusion protein measured in the expression solution was 12500units / mL (1.5×10 6 cells / mL). The activity in the frozen lysed cell extract is only 900units / 1.5×10 6 cells. The expression level of the fusion protein secreted in the cult...

1. Technical field

[0001] The invention belongs to the technical field of polypeptide medicine prepared by genetic engineering. 2. Background technology

[0002] Stem cell factor (SCF, also called Kit ligand, Steel factor, mast cell growth factor, etc.) is a multifunctional cytokine that can cooperate with other cytokines at the multi-level hematopoietic level to promote hematopoietic stem / progenitor cells and various blood cells. It can also maintain the survival and proliferation of melanocytes, blast cells and mast cells, and promote their proliferation and differentiation. Its target cells include pluripotent hematopoietic stem / progenitor cells, erythroid cells, megakaryocytes, monocyte-macrophages, some lymphoid precursor cells, mast cells, melanocytes and blast cells.

[0003] Natural SCF is a kind of glycoprotein, which is mainly expressed by stromal cells, fibroblasts and endothelial cells, and the ratio of membrane-bound SCF and soluble SCF expression varies greatl...

Examples

specific Embodiment approach 2

[0030] Steps 1 and 2 are the same as steps 1 and 2 in Embodiment 1

[0031] 3. With a doubling time of 19 hours, living cells account for 95%, and a density of 1.5×10 6 Cells / mL 40 mL of Sf9 suspension cells cultured in SF900II medium containing 4% newborn bovine serum were centrifuged at 500 rpm for 10 minutes, and the collected cells were resuspended in 40 mL of SF900II serum-free medium. According to the multiplicity of infection (MOI) = 0.1, the titer was 1.0×10 7 0.6 ml of the recombinant virus AcNPV-rhdSCF stock solution per ml. The rotating speed of the stirring blade was 60 rpm, and after culturing at 27° C. for 48 hours, centrifuge at 10,000 rpm for 5 minutes, discard the cells and collect 40 ml of the supernatant. The activity of the fusion protein measured in the expression solution was 12500units / mL (1.5×10 6 cells / mL). The activity in the frozen lysed cell extract is only 900units / 1.5×10 6 cells. The expression level of the fusion protein secreted in the cult...

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