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Human parathyrine PTH (1-34) fusion protein and use thereof

A parathyroid hormone and fusion protein technology, applied in the field of medical bioengineering, can solve the problems of difficult and difficult to solve the embedding process, and achieve the effects of long-term biological activity, increase biological activity, and prolong circulation half-life.

Inactive Publication Date: 2017-01-04
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve the above problems, researchers have studied the PTH (1-34) dosage form and developed a PTH (1-34) sustained-release microsphere preparation (such as CN 103157096A), but the drug embedded in the sustained-release microsphere The process is difficult, especially in mass production, this problem is more difficult to solve

Method used

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  • Human parathyrine PTH (1-34) fusion protein and use thereof
  • Human parathyrine PTH (1-34) fusion protein and use thereof
  • Human parathyrine PTH (1-34) fusion protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the construction of fusion protein and its plasmid

[0039] First, an RNA synthesis company is commissioned to synthesize RNA, and then cDNA is synthesized from RNA using reverse transcriptase. Then different primers were used to amplify the desired PTH (1-34) fragment by polymerase chain reaction (PCR). The Fc fragment of immunoglobulin is obtained by PCR amplification from the cloned IgG1 Fc plasmid. Finally, PCR is used to fuse the PTH(1-34) fragment and the Fc sequence, thereby constructing the DNA sequence of the PTH(1-34)-Fc fusion protein.

[0040] The polymerase chain reaction included: denaturation at 95°C for 30 seconds; annealing at 56°C for 45 seconds; extension at 72°C for 2 minutes, 30 cycles. Using TA cloning kit, clone the PCR products of PTH(1-34) and Fc into pET-32a plasmid respectively, transfect, E.coli BL21(DE3), select white colonies, add LB medium, and culture overnight. Then the plasmid was extracted with Qiangen plasmid extracti...

Embodiment 2

[0054] Example 2: Expression of PTH(1-34)-Fc fusion protein in cells

[0055] This example provides an example of expressing PTH(1-34)-Fc fusion protein by taking mammalian cells as an example. Three fusion proteins of P1, P2 and P3 were cloned into pET-32a plasmid (Invitrogen). A human F1t-1 signal peptide sequence was added to the 5' end of the fusion protein. The above three PTH(1-34)-Fc high-purity plasmid DNAs were extracted with a plasmid DNA purification kit (MoBio Company). Then, the plasmid DNA was introduced into CHO cells by using the FUGEN6 transfection kit (ROCHE Company). We compared the expression levels of these four PTH(1-34)-Fc fusion proteins by means of transient transfection. CHO cells were cultured in cell culture dishes with DMEM complete medium containing 10% fetal bovine serum. When the cells grew to a certain point, the complex of plasmid DNA and FUGEN6 reagent (Roche) was added to the cell culture medium, and the supernatant was collected after c...

Embodiment 3

[0056] Example 3: Establishment of high-efficiency expression cell lines

[0057] After confirming that the P3 fusion protein is the best molecular combination, stable and high-efficiency expression cell lines of CHO were established by means of stable transfection and gene amplification. The gene containing the P3 fusion protein was cloned into a plasmid containing the DHFR gene. High-purity plasmid DNA was extracted with a DNA purification kit from MoBio, and then transfected into CHO cells by electrodrilling. The transfected cells were cloned in selective culture to obtain resistant clones. Gene amplification was carried out under pressure with methotrexate (MTX) to obtain highly expressed cell lines. Finally, the cells are expanded to produce the fusion protein on a large scale in a bioreactor. The concentration of fusion protein was determined quantitatively by ELISA. Using this method, the P3 fusion protein was successfully produced on a large scale. The output can ...

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Abstract

The invention belongs to the technical field of medical bioengineering and discloses a human parathyrine PTH (1-34) fusion protein and its use in preparation of a pharmaceutical composition. A human IgG-derived Fc zone is connected to a C end of PTH (1-34) so that a circulating half life of the PTH (1-34) is effectively prolonged and / or biological activity of the PTH (1-34) is improved and the existing PTH (1-34) degradation problems are solved. The human parathyrine PTH (1-34) fusion protein has long-acting biological activity, has a high expression level and can be used for industrial large-scale production.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a human parathyroid hormone PTH (1-34) fusion protein and its application in the preparation of pharmaceutical compositions. Background technique [0002] Osteoporosis is the most common metabolic bone disease characterized by low bone mass and degeneration of bone tissue. Most of the drugs on the market for the treatment of osteoporosis, such as estrogen, bisphosphonates and calcitonin, are bone resorption inhibitors. Other drugs, such as fluoride and parathyroid hormone (PTH), are bone formation promoters. [0003] PTH (1-34) (teriparatide), whose trade name is Forteo, was developed by Eli Lilly and Company. It was first listed in the United States in December 2002, and then successively sold in Austria, Denmark, Finland, Germany, Greece, Iceland, Ireland, the Netherlands, Listed in Norway, Portugal, Switzerland, UK, Australia and other countries. This product...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/11C12N15/63C12N5/10C12N1/21C12N1/19C12P21/02A61K38/29A61K47/48A61K48/00A61P19/10
Inventor 康旭陈军陶安进袁建成
Owner HYBIO PHARMA
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