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Fusion protein with high-efficient platelet-increasing role and preparation method thereof

A technology of fusion protein and platelet raising, which is applied in the fields of biotechnology and genetic engineering pharmacy, can solve the problems of ineffective activation, low bioavailability, large toxic and side effects, etc., and achieves the effect of reducing the treatment cycle, reducing the treatment time, and achieving a remarkable effect.

Active Publication Date: 2012-03-21
ARMY MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since a single TPO mimetic peptide can only bind to one c-Mpl, it cannot effectively activate its receptor signaling pathway, so that the rTMP-GH fusion protein mainly exerts the effect of increasing platelets through human growth hormone molecules. , more toxic and side effects

Method used

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  • Fusion protein with high-efficient platelet-increasing role and preparation method thereof
  • Fusion protein with high-efficient platelet-increasing role and preparation method thereof
  • Fusion protein with high-efficient platelet-increasing role and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Recombinant expression plasmid pBV220 / TMP 2 - Construction and identification of hGH

[0057] (1) First obtain the gene sequence encoding a fusion protein (TMP-hGH) of a TPO mimetic peptide and human growth hormone:

[0058] Using the plasmid pMD-hGH containing human growth hormone (preserved in our laboratory, for its construction method, refer to the invention patent of application number 200510057217.7) as a template, the DNA sequence encoding TPO mimetic peptide and bridging peptide was loaded into the N- end. The PCR primer sequences used are as follows:

[0059] P1: 5′-att gag gga cca aca tta cgt cag tgg ctg gct gca cgt gct ccaggt tct ggt tct ggt ttc cca act att cca ctg agt c-3′

[0060] P2: 5′-gca g gt cga c at tta gaa gcc aca cga ccc ttc-3′

[0061] In P1, the bold part is the nucleotide sequence encoding the TPO mimetic peptide (SEQ ID NO1 in the sequence table), the italic part is the nucleotide sequence encoding the bridging peptide Pro-Gly-S...

Embodiment 2

[0069] Example 2: TMP 2 -Expression and purification of hGH fusion protein in Escherichia coli

[0070] (1) High expression of TMP 2 - Positive strain screening for hGH fusion protein:

[0071] Pick at least 10 transformants with pBV220 / TMP 2 - DH5α Escherichia coli single clone of hGH recombinant plasmid, inoculated in a shaking tube containing 2ml of LB medium, cultured overnight on a shaker at a constant temperature at 32°C, and inoculated 100ul overnight bacteria into fresh LB medium at a ratio of 1:10 the next day In 32°C constant temperature shaker shaking culture for 24 hours, then change the temperature to 42°C for induction expression for 5 hours. Centrifuge at 12000rpm for 2 minutes to collect bacteria, perform high-pressure homogenization at 700MPa to break bacteria, centrifuge at 12000rpm for 20 minutes to extract inclusion body protein, wash inclusion body protein with inclusion body washing solution, and then dissolve inclusion body with 20mM TRIS buffer (PH8....

Embodiment 3

[0074] Example 3: TMP 2 - Determination of platelet-increasing activity of hGH fusion protein

[0075] (1) Copying the experimental animal model of thrombocytopenia mice: taking BALB / c mice as the experimental subjects, 60 Coγray 5Gy whole body irradiated experimental mice at one time, and replicated the animal model of thrombocytopenia induced by radiation injury (this situation is common in acute radiation sickness and thrombocytopenia induced by tumor radiotherapy).

[0076] (2) Experimental grouping: experimental animals are divided into three groups

[0077] The first group was given recombinant TMP after irradiation 2 - hGH fusion protein therapy, 100ug / kg / d, subcutaneous injection, continuous use for 7 days;

[0078] The second group was treated with recombinant TMP-hGH after irradiation, 100ug / kg / d, subcutaneous injection, for 7 consecutive days;

[0079] The third group is the normal saline control group.

[0080] (3) Methods and results: 7 days after irradiation...

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Abstract

The invention relates to a fusion protein with high-efficient platelet-increasing role, and the fusion protein consists of two TPO mimetic peptides which are connected in series and a human growth hormone molecule. The fusion protein can significantly improve the bioavailability in the aspect of platelet generation, reduce the using quantity and reduce the toxicity or the side effects. The fusionprotein can greatly reduce the treatment cycle and the treatment time, and the platelet-increasing effect of the fusion protein is more significant. The fusion protein can be used for producing drug preparations to treat a variety of primary or secondary thrombocytopenia and other diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering pharmacy, and specifically relates to a fusion protein capable of promoting platelet production and having a high-efficiency platelet-increasing effect and a preparation method thereof. Background technique [0002] Platelets play an important role in the life activities of the body. Thrombocytopenia can cause bleeding, infection, and even life-threatening. With the development of society, more and more patients with thrombocytopenia are seen, and the data show that the occurrence of most thrombocytopenia is due to thrombocytopenia. The precursor of platelets is megakaryocytes, and its generation process follows the typical staged differentiation pattern of hematopoietic stem cells: bone marrow hematopoietic stem cells-early myeloid common progenitor cells-megakaryotic / erythroid progenitor cells-megakaryotic progenitor cells-megakaryocytes-platelets. It can be seen that the pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/70C12N15/81A61K38/27A61K38/16A61P7/00
Inventor 王军平粟永萍许杨王崧申明强陈芳陈默
Owner ARMY MEDICAL UNIV
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