Preparation and application of dimerized fusion protein

A fusion protein and doublet technology, applied in the direction of albumin peptide, fusion polypeptide, serum albumin, etc., can solve the problems of neutralizing antibody, failure to detect thrombopoietic activity, and inhomogeneous target

Active Publication Date: 2014-09-17
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the target substances will increase antigenicity after being mutated. After such drugs are injected into the human body, they will lead to the production of neutralizing antibodies
The second method is the chemical modification method. In this regard, although long-acting drugs such as polyethylene glycol-modified interferon have been marketed, the problem of heterogeneity of the target object caused by the modification of such macromolecular compounds is currently difficult to solve. Solve (Jiang He and Gong Mengjia, 2012)
However, when we injected this fusion protein subcutaneously into normal mice, its pro-thrombocytogenic activity could not be detected, and it had no effect in the essential thrombocytopenia model

Method used

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  • Preparation and application of dimerized fusion protein
  • Preparation and application of dimerized fusion protein
  • Preparation and application of dimerized fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0075] Example 1 Construction and Screening of Dimerized Thrombopoietin Mimetic Peptide TMP Duplex-Human Serum Albumin Fusion Protein CHO Expression Vector

[0076] One. Construction of the vector comprising TMP-L1-TMP-L5-H-fip-HSA

[0077] Cloning of the fragment of HSA cDNA: HSA cDNA was amplified in two stages from the plasmid pcDNA3.1-fip-HSA (the full gene sequence of HSA was synthesized by Bao Biological Company, and the pcDNA3.1 plasmid was purchased from Invitrogen) by PCR method, respectively. It is fip(Post1)-HSA(Pre1) (fip is a peptide segment selected from the fip domain, HSA is human serum albumin, Pre represents the first half, post represents the second half, and the gene sequence is shown in Seq ID No: 9, The amino acid sequence is shown in Seq ID No: 10) and HSA(Post1)-His (Post is the back end of HSA, His is the tail mark of histidine, and the gene sequence of HSA(Post1)-His is shown in Seq ID No: 11 , the amino acid sequence is shown in Seq ID No: 12). The...

Embodiment 4

[0124] Example Dimerized Thrombopoietin Mimetic Peptide TMP Duplex-Human Serum Albumin Fusion Protein Expression in Mammalian Cell CHO

[0125] A large number of plasmid extraction kits (Qiagen, Shanghai)

[0126] L-glutamine, sodium hypoxanthine and thymine supplementation reagent (HT) were purchased from Invitrogen, USA;

[0127] T75 cell culture flasks were purchased from Denmark NUNC Company;

[0128] 2-L WAVE cell culture bags were purchased from the Medical Group Department of General Electric Company of the United States;

[0129] Chinese hamster ovary suspension cells (CHO-S) were purchased from Invitrogen Corporation of the United States, and the cells were a clone isolated from Chinese hamster ovary subline clone cells (CHO-K1). The CHO-S parental cell line was chosen for cell culture and transfection. CHO-S cells are suitable for serum-free suspension culture using FreeStyle Supplementation Reagent (HT) supplemented with 8mM L-glutamine and 2mM Thymine TM CHO s...

Embodiment 1

[0133] Clones No. 2-8 obtained in Example 1 were also transiently transfected into CHO-S cell lines by the same method, and respectively obtained TMP-L2-TMP-L5-H-fip-HSA sequence, TMP-L2-TMP -L5-H-fip-HSA sequence, TMP-L2-TMP-L5-H-fip-HSA sequence, HSA-L6-H-L7-TMP-L1-TMP sequence, HSA-L6-H-L7-TMP- L2-TMP sequence, HSA-L6-H-L7-TMP-L3-TMP sequence, HSA-L6-H-L7-TMP-L4-TMP sequence encoding target product supernatant cells transient expression.

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Abstract

The invention discloses preparation and application of a dimerized thrombopoietin (TPO) mimic peptide TMP diad-human serum albumin fusion protein. The fusion protein contains an HAS and two TPO mimic peptides which are connected through a linkage oligopeptide and a dimerization domain so as to ensure the fusion protein is dimerized correctly when expression. The two TPO mimic peptides can be connected with N terminal or C terminal of HAS. The dimerized HAS protein carrier and the TPO mimic peptide fusion protein provided by the invention have a significant activity of promoting thrombopoiesis in vivo and can be applied in preparation of drugs for treating primary and secondary thrombocytopenia diseases.

Description

technical field [0001] The invention belongs to the field of genetic engineering pharmacy, and specifically relates to a dimerized thrombopoietin mimetic peptide TMP duplex-human serum albumin fusion protein and a preparation method and application thereof. Background of the invention [0002] The half-life of most therapeutic proteins and peptides is generally short (Jin Guangguang et al., 2010), requiring frequent injections; and the price of such marketed drugs is relatively high. This brings unbearable physical, psychological and economic burdens to patients who need long-term application of therapeutic proteins and peptides. Therefore, protein engineering to prolong the half-life of these therapeutic proteins and active polypeptides has become a research hotspot, and the development of long-acting recombinant proteins and active polypeptide drugs has also become an important direction for the secondary development of first-generation genetic engineering products ( Zhao...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/19A61K47/48A61P7/04
CPCA61K38/00A61K38/16C07K19/00A61P7/04C07K14/524C07K14/765C07K2319/00C07K2319/70
Inventor 不公告发明人
Owner LANZHOU UNIVERSITY
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