Tranduced peptide-huamn erythrogenin fusion protein and its medicinal composition

A technology of erythropoietin and fusion protein, applied in the field of fusion protein, can solve the problem of high production cost, achieve the effects of low cost, improved transduction efficiency and simplified purification process

Inactive Publication Date: 2007-11-21
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] (2) High production cost
Therefore, the currently produced commercial EPO is produced by large-scale mammalian cell culture, and the production cost is high.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Extraction of Human Fetal Liver Total RNA

[0030] The RNAgentsR Total RNA Isolation System from Promega was used to extract total RNA from induced fetal liver.

Embodiment 2

[0031] Cloning of embodiment 2 hEPO gene

[0032] Design primers with reference to the EPO cDNA sequence in Genebank:

[0033] Primer 1: 5'-GGAATTCGCACCACCACGCCTAATC-3' (SEQ ID NO: 7)

[0034] Primer 2: 5'-GTCGACTCATCATCTGTCCCCTGTCCT-3' (SEQ ID NO: 8)

[0035] Using the RNA in Example 1 as a template, PCR amplification was performed according to the protocol recommended by the kit manufacturer to obtain full-length hEPO cDNA, with EcoRI and Sal I sites at the 5' and 3' ends, respectively.

[0036] Reverse transcription and PCR amplification: the reverse transcription PCR kit produced by Perkin ElmerCetus was used. Take 1-2ug of the extracted total cellular RNA, add it to a 20ul reverse transcription system, use oligo dT as a primer, and act at 42°C for 1h to synthesize the first strand of cDNA. The reverse transcription reaction was terminated at 99°C for 5 min. Add 20ul of the reverse transcription product into a 100ul PCR reaction system, which contains upstream and down...

Embodiment 3

[0037] Example 3 Construction and Identification of Recombinant hEPO Expression Plasmid

[0038] The RT-PCR product of hEPO in Example 2 was double digested with EcoRI and SalI, and then ligated with the expression vector pBV220 fragment recovered by the same digestion at a molar ratio of 4:1, transformed into competent Escherichia coli DH5α, and positive recombinants were screened pBV hEPO. Sent to Shanghai Shengong Company for sequencing.

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PUM

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Abstract

A transduction peptide-human hematopoietin fusion protein, nucleic acid molecule of nucleic acid sequence for encoding fusion protein, expression carrier containing the nucleic acid molecule and its usage of fusion protein in preparation of medicine are disclosed. It can be used for hemorrhagic anemia, anemia after surgery, cancer relative anemia, human immune-deficient viral infection, hyperosteogeny abnormal syndrome, premature birth anemia, renal anemia, bone marrow transplantation, auto-blood collection, gravida anemia, hemoglobin disease and aregenerative treatments.

Description

field of invention [0001] The invention relates to a fusion protein with medicinal value, specifically a transduction peptide-human erythropoietin fusion protein, and a nucleic acid molecule encoding the fusion protein. The present invention also relates to pharmaceutical compositions containing the fusion protein. Background of the invention [0002] In 1906, Carnot discovered that there are factors regulating hematopoiesis in the blood of anemic animals. In 1948, Bonsdloff et al. proposed that this hematopoietic factor is erythropoietin (erythropoietin, erythropoietin, EPO). In 1957, Jacobson et al confirmed that EPO comes from the kidney, and the concept of EPO has been generally accepted since then. After successfully cloning the human EPO gene in 1985, genetic engineering began to produce recombinant human EPO in large quantities, and it was widely used clinically. EPO treatment of renal anemia has been recognized by the world, with the in-depth study of EPO clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61K9/02A61K9/06A61K9/12A61P7/06C12N15/62C12N15/63
Inventor 李前孙曼霁
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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