Fusion protein of thrombopoietin mimetic peptide (TMP) diad and human serum albumin (HSA), and preparation method and application thereof

A technology of thrombopoietin and human serum albumin, applied in the field of biotechnology and genetic engineering pharmaceuticals, can solve the problems of short biological half-life, unsatisfactory application effect, small molecular weight, etc., and achieve the effect of prolonging the half-life

Inactive Publication Date: 2012-04-11
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the small molecular weight of the TMP dyad polypeptide, the biological half-life in vivo is very short, so the in vivo application effect is not ideal

Method used

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  • Fusion protein of thrombopoietin mimetic peptide (TMP) diad and human serum albumin (HSA), and preparation method and application thereof
  • Fusion protein of thrombopoietin mimetic peptide (TMP) diad and human serum albumin (HSA), and preparation method and application thereof
  • Fusion protein of thrombopoietin mimetic peptide (TMP) diad and human serum albumin (HSA), and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: the total gene synthesis of human serum albumin HSA

[0068] 1. First, optimize the gene sequence encoding HSA (including signal peptide and propeptide consisting of 24 amino acid residues) according to the preferred codons of Pichia pastoris. The optimized full-length gene sequence of HSA is shown in SEQ ID NO: 10 The 1-72 bp at the 5' end is the base sequence encoding HSA signal peptide and propeptide. The signal peptide and propeptide are automatically excised during expression and secretion in yeast cells.

[0069] 2. Entrust Shanghai Sangon Bioengineering Company to synthesize the optimized full-length gene of HSA and load it into pUC57 plasmid (the pUC57 plasmid vector is provided by Shanghai Sangon Biotechnology Company) to obtain pUC57-HSA.

Embodiment 2

[0070] Example 2: pPICZα / HSA-L-(TMP) 2 Construction of recombinant expression vector

[0071] 1. Code HSA-L-(TMP) 2 Obtaining of cDNA fragments

[0072] (1) Design and synthesize PCR primers:

[0073] P1 (SEQ ID NO: 7): 5'-agc ttc gaa acg_atg aag tgg gta acc ttt att tcc ctt c 3′

[0074] P2 (SEQ ID NO: 8): 5'-ggc tct agc agc caa cca ttg tct caa agt tgg acc ctc aat acc aga tcc acc gcc tcc aga tcc acc tcc gcc gga tcc taa gcc taa ggc agc ttg ac-3'

[0075] P3 (SEQ ID NO:9): 5'-at cgc ggc cgc tta tgc tct agc tgc aag cca ctg tct cag ggt agg tcc ctc gat tgg acc aga tgg ggc tct agc agc caa cca ttg-3′

[0076] Among them, the base underlined in P1 is the recognition site sequence of restriction endonuclease Asu II; the 5' end of P2 is the sequence encoding TMP, the italic part in the middle is the Linker sequence between HSA and TMP, and the 3' end is the HSA gene C-terminal specific recognition sequence; the base underlined in P3 is the restriction endonuclease Not I recog...

Embodiment 3

[0082] Example 3: HSA-L-(TMP) 2 Expression and purification of fusion proteins

[0083] Take the integrated pPICZα / HSA-L-(TMP) frozen at -70℃ 2 The engineered yeast strains were inoculated on the YPDS plate at 30°C for overnight culture and activation, and a single colony with a good appearance was selected and inoculated in the BMGY medium, and cultured at 220rpm on a constant temperature shaker at 30°C for 16-18 hours to OD 600 ≈3~5, then transfer to OD once at 1:10 600 It is about 4, and the bacterial liquid is used as the seed liquid. Then the seed solution was transplanted into a 15L fermenter (Bio Company, Switzerland) equipped with 7L basic salt medium for high-density culture and fermentation. Each liter of basal medium contains 50g of glycerol, 12ml of concentrated phosphoric acid, 2.6g of KOH, CaSO 4 2H 2 O 0.6g, K 2 SO 4 9.5g, MgSO 4 ·7H 2 O 7.8g, biotin 0.32mg, YTB solution (containing 65g / L of FeSO 4 ·7H 2 O, 6g / L CuSO 4 ·5H 2 O, 20g / L ZnSO 4 ·7H 2...

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Abstract

The invention discloses a fusion protein of a thrombopoietin mimetic peptide (TMP) diad and a human serum albumin (HSA), and a preparation method and application thereof. The fusion protein comprises one HSA and two TMPs, wherein the HSA is positioned at the N-terminal of the fusion protein, and the two TMP sequences are connected in series to form a diad which is positioned at the C-terminal of the fusion protein; or the HSA is positioned at the C-terminal of the fusion protein, and the two TMP sequences are connected in series to form a diad which is positioned at the N-terminal of the fusion protein. Connecting peptides are arranged between the HSA and the TMP diad as well as between the two TMPs. The fusion protein has a characteristic of obviously promoting thrombocytopoiesis, has a long half life in vivo and can be used for the preparation of medicaments for treating multiple primary or secondary thrombocytopenia and other diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering pharmacy, and in particular relates to a fusion protein of thrombopoietin mimetic peptide duplex and human serum albumin as well as its preparation method and application. Background technique [0002] In clinical practice, primary and secondary thrombocytopenia caused by various reasons are often encountered, such as primary thrombocytopenic purpura, aplastic anemia, and thrombocytopenia caused by tumor chemotherapy / radiotherapy. For such diseases, it is more suitable to use long-acting platelet-increasing drugs for treatment. On the one hand, it can reduce the number of medications and reduce the pain of acupuncture for patients; on the other hand, it can reduce the dosage of drugs and reduce the cost of treatment. [0003] Platelets are produced by megakaryocytes, and promoting the proliferation and differentiation of megakaryocytes is the main means to increase the level of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/63C12N1/19C12N1/21C12N5/10A61K38/38A61K38/10A61P7/04C12R1/19C12R1/84
Inventor 王军平申明强许杨陈芳陈默王崧王艾平粟永萍
Owner ARMY MEDICAL UNIV
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