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Recombinant human platelet auxin/dry cell factor fusion protein and preparation thereof

A technology of thrombopoietin and stem cell factor, which is applied in the fields of peptide/protein components, peptide preparation methods, recombinant DNA technology, etc., can solve problems such as unsatisfactory effect and short platelet stability time

Inactive Publication Date: 2006-05-31
NANJING UNIV
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  • Abstract
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Problems solved by technology

Although rhTPO can quickly increase the level of platelets in the body, the stable time of platelets is not long, and the effect is not ideal

Method used

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  • Recombinant human platelet auxin/dry cell factor fusion protein and preparation thereof
  • Recombinant human platelet auxin/dry cell factor fusion protein and preparation thereof
  • Recombinant human platelet auxin/dry cell factor fusion protein and preparation thereof

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Experimental program
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Effect test

Embodiment Construction

[0059] 1. Use pUC18-SCF as template, use primers 1 and 2 to synthesize SCF gene fragment by PCR method and clone it into pBluscript plasmid vector to obtain pBluscript-3'SCF plasmid, then use pUC18-TPO as template, use primers 3 and 4 The TPO gene fragment was synthesized by PCR method and cloned into pBluscript-3'SCF plasmid vector to obtain pBluscript-TS plasmid. Sequencing proved that the result was completely consistent with the designed sequence. (See attached for the process figure 1 )

[0060] 2. Digest the pBluscript-TS plasmid to obtain the TPO / SCF gene fragment, and insert it into the AcNPV transfer vector pAcSecG2T to obtain the recombinant transfer vector pAcSecG2T-TS. Sf9 was co-transfected with pAcSecG2T-TS and AcNPV DNA, and the recombinant virus AcNPV-rhTPO / SCF without wild-type virus was selected by screening and assay.

[0061] 3. Take the doubling time as 21 hours, live cells account for 90%, and the density is 6×10 5 Cells / mL single-layer Sf9 cells were...

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Abstract

The invention is the gene engineering preparation polypeptide remedy technology field of biology technology. The aid is to build a double function fusion albumen gene of rhTPO / SCF, express and prepare with more biology activityú¼less side-effect rhTPO / SCF in insect cell. The gene of rhTPO / SCF includes 1-157 amino acids coding sequence with TPO, 16 amino acids continual peptide coding sequence, 1-145 amino acids coding sequence of SCF and codoní»s DNA fragments. By taking the rhTPO / SCF into TF1 cells experiment, which preparation from the infect recombination virus growing in adherence S19 cell, it mensurates that the specific activity is 2.0 í‡10 to the power 5 units / mg; by Mo7e cells experiment, the specific activity is 8.3í‡10 to the power 5 units / mg. It can be used to cure low blood platelet cause by radiotherapy, chemotherapy, bone marrow transplantationíú

Description

1. Technical field [0001] The invention belongs to the technical field of polypeptide medicine prepared by genetic engineering. 2. Technical background [0002] Platelets are the key components involved in blood coagulation. Hematopoietic stem cells first undergo gradual differentiation into megakaryotic progenitor cells, then differentiate into mature megakaryocytes, and finally lyse to produce platelets. This complex differentiation and maturation process is regulated by many hematopoietic factors, and is also affected by the interaction between hematopoietic stem cells, extracellular matrix and surrounding bone marrow cells. At present, it is agreed that Thrombopoietin (TPO) is the most important cytokine that regulates the generation and proliferation of megakaryocytes. It works together with IL-3 and SCF to promote the differentiation of hematopoietic stem cells into oriented megakaryocytes, and then in TPO, IL-3, SCF, IL-6, IL-11 and other factors participate in the d...

Claims

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Application Information

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IPC IPC(8): C12N15/62A61K38/17C07K1/14C07K19/00C12N15/866C12P21/02
Inventor 秦浚川臧宇辉朱洁袁达文
Owner NANJING UNIV
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