Recombinant human erythropoietin-CTP fusion protein production process and application

A technology of fusion protein and -CTP, which is applied in the direction of drug combination, peptide/protein composition, recombinant DNA technology, etc., can solve the problems of frequent administration of patients and short half-life of EPO in vivo

Active Publication Date: 2013-03-27
哈药集团股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the demand for EPO in my country is increasing year by year, but ordinary EPO has problems such as short half-life in vivo and frequent administration of patients.

Method used

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  • Recombinant human erythropoietin-CTP fusion protein production process and application
  • Recombinant human erythropoietin-CTP fusion protein production process and application
  • Recombinant human erythropoietin-CTP fusion protein production process and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1 expression strain construction method

[0040] The EPO-CTP fusion protein gene that will be connected on the pMD18-T carrier, utilizes Not I and EcoR I double enzyme digestion to identify positive clone ( figure 1 ), sequenced. After obtaining positive clones with the correct sequence, the fusion gene was connected to the pcDNA eukaryotic expression vector, and the constructed expression strain could be transfected into CHO cells after extracting the plasmid. Schematic diagram of the structure of the EPO-CTP fusion protein, see figure 2 .

[0041] Get the plasmid of EPO-CTP fusion gene, use Not I and EcoR I to carry out double enzyme digestion ( image 3 ), recover the small fragments, and take the pcDNA eukaryotic expression vector at the same time, use Not I and EcoR I to carry out double enzyme digestion to recover the large fragments, connect the recovered two fragments with T4 ligase, transform them into E. coli DH5α, and screen for positive recomb...

Embodiment 2

[0042] Example 2 EPO-CTP fusion protein transfection of CHOK1 cell line

[0043] Transform the recombinant expression plasmid into a mammalian host cell line to express the EPO-CTP fusion protein. For high level stable expression, the preferred host cell line is CHOK1. The present invention adopts electrotransformation, 150 μg linearized DNA is added to 5 μl salmon sperm DNA, CHOK1 host cells are 5×106, after mixing, 180V electric shock is used twice, and the cell suspension after electrotransformation is spread into a plate with medium. When clonal clusters appeared in the plate, single clones were picked and expanded for culture. After 3 times of clonal screening, a relatively stable high-expressing cell line was obtained.

Embodiment 3

[0044] Example 3 Large-scale, high-density, carrier-attached perfusion culture of animal cells

[0045] The animal cell microcarrier-attached perfusion culture method mainly includes the inoculation of cells, the initial growth stage, changing the culture conditions, the subsequent growth stages and monitoring the culture conditions. The specific steps are as follows:

[0046] 1. Inoculation of cells.

[0047] A cell line expressing EPO-CTP was resuscitated in the cell bank, and passed three times in a T25 square bottle with DMEM medium plus 10% fetal bovine serum to adjust the cell state. When the cells are in the logarithmic growth phase, digest with trypsin, add serum to stop the digestion, and add 10% fetal bovine serum in DMEM medium for seed strand amplification, cell expansion according to T25-T75-T150 square flask- The order of the 2L spinner bottle was passed down step by step. Bioreactors with microcarriers were inoculated with spinner bottle digested cells. The c...

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Abstract

The invention discloses a recombinant human erythropoietin-CTP fusion protein production process. The process is characterized in that a microcarrier suspension perfusion culture technology or a serum-free suspension fed-batch culture technology is adopted as a recombinant engineering cell line culture manner. The invention also discloses a recombinant human erythropoietin-CTP fusion protein purifying process. Through optimization, a simple process flow is obtained, such that high-purity and high-activity recombinant human erythropoietin-CTP fusion protein is obtained. The invention also relates to the ticokinetics study of the recombinant human erythropoietin-CTP fusion protein in monkey bodies, and the application of the recombinant human erythropoietin-CTP fusion protein in treating rat renal anemia.

Description

technical field [0001] The invention belongs to the field of biopharmaceutical technology. It specifically relates to a production process of a fusion protein of recombinant human erythropoietin and human chorionic gonadotropin beta subunit carboxyl terminal peptide, including the acquisition of recombinant engineered cell lines of the fusion protein, cell culture and protein purification The technical method and the application of the recombinant human erythropoietin-CTP fusion protein in the pharmacokinetic experiment of monkeys and the treatment of renal anemia in rats. Background technique [0002] EPO is a glycoprotein mainly produced by the kidney with a molecular weight of about 35KD. It is the main hormone regulating erythroid hematopoiesis in the human body and plays an important regulatory role in the proliferation, differentiation and maturation of erythroid progenitor cells. At present, the demand for EPO in my country is increasing year by year, but ordinary EP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C07K19/00A61K38/24A61K38/18A61P7/06
Inventor 王锐张秀芹孟庆勇董敏刘铁成梁秋波李会成
Owner 哈药集团股份有限公司
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