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Fusion protein of human parathyrine (1-34) diad and human seralbumin and preparation thereof

A technology of human serum albumin and parathyroid hormone, applied in the field of DNA recombination, can solve the problems of limited application and high price, and achieve the effect of avoiding the problems of fusion protein stability and immunogenicity

Inactive Publication Date: 2009-10-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high price and the inconvenience caused by frequent medication severely limit the application of this drug

Method used

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  • Fusion protein of human parathyrine (1-34) diad and human seralbumin and preparation thereof
  • Fusion protein of human parathyrine (1-34) diad and human seralbumin and preparation thereof
  • Fusion protein of human parathyrine (1-34) diad and human seralbumin and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Cloning of hPTH(1-34) doublet cDNA;

[0034]Use a plasmid extraction kit to extract the plasmid in JM109 / pPIC9K-PTH(1-84)-HSA, and use the extracted plasmid as a template to obtain the first hPTH(1-34) cDNA, namely PTHa', by PCR amplification , the primers used are as follows:

[0035] Upstream primer P1: 5'-TACTATTGCCAGCATTGCTGC-3'

[0036] Downstream primer P2: 5'-GGAAACGGAGAAGTTGTGAACGTCTTG-3'

[0037] The PCR reaction system is: pPIC9K-PTH(1-84)-HSA 1μl, 10μmol / L primers 1μl each, 2.5mmol / L dNTP 4μl, 10×pyrobest Buffer 5μl, 5U / μl Pyrobest DNA polymerase 0.5μl , add double distilled water to make up 50μl.

[0038] The conditions of the PCR reaction were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 20 seconds, and 35 cycles; final extension at 72°C for 7 minutes.

[0039] The upstream primer is the universal primer α-Factor on pPIC9K. The PCR product obtained in thi...

Embodiment 2

[0055] Embodiment 2: the cloning of HSA cDNA;

[0056] HSA cDNA was amplified from the pPIC9K-PTH(1-84)-HSA plasmid by PCR, and the primers used were as follows:

[0057] Upstream primer P5: 5'-GATGCACACAAGAGTGAG-3'

[0058] Downstream primer P6: 5'-ATTTGCGGCCGCTTATAAGCCTAAGGCAGCTTG-3'

[0059] The PCR reaction system is: pPIC9K-PTH(1-84)-HSA 1μl, 10μmol / L primers 1μl each, 2.5mmol / L dNTP 4μl, 10×Pyrobest Buffer 5μl, 5U / μl Pyrobest DNA polymerase 0.5μl , add double distilled water to make up 50μl.

[0060] The conditions of the PCR reaction were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 45 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 2 minutes, and 30 cycles; final extension at 72°C for 10 minutes.

[0061] Use 0.7% agarose gel to analyze the product of overlapping PCR reaction, use 1Kbp DNA Ladder Marker as a standard, and use PCR gel recovery kit to recover the target fragment of 1800bp in the loading lane, which is HSA cDNA.

...

Embodiment 3

[0063] Example 3: Cloning of hPTH(1-34) doublet cDNA and HSA cDNA fusion gene;

[0064] PCR amplification of hPTH(1-34) doublet cDNA, primers are as follows:

[0065] Upstream primer P1: 5'-TACTATTGCCAGCATTGCTGC-3'

[0066] Downstream primer P7: 5'-CTCACTCTTGTGTGCATC-3'

[0067] The PCR reaction system is: T-PTH plasmid 1 μl, 10 μmol / L primers 1 μl each, 2.5 mmol / L dNTP 4 μl, 10×PyrobestBuffer 5 μl, 5U / μl Pyrobest DNA polymerase 0.5 μl, add double distilled water to make up 50 μl.

[0068] The conditions of the PCR reaction were: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 20 seconds, and 30 cycles; final extension at 72°C for 7 minutes.

[0069] For PCR amplification of HSA cDNA, the primers are as follows:

[0070] Upstream primer P5: 5'-GATGCACACAAGAGTGAG-3'

[0071] Downstream primer P6: 5'-ATTTGCGGCCGCTTATAAGCCTAAGGCAGCTTG-3'

[0072] The PCR reaction system is: T-HSA plasmid 1 ...

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Abstract

The invention relates to a fusion protein of human parathyrine (1-34) diad and human seralbumin and a preparation technology thereof, belonging to the technical field of long lasting recombinant fusion protein. The fusion protein comprises the first region of the diad which has at least 85% homologous sequence with the human parathyrine (1-34) and the second region which has at least 85% homologous sequence with the human seralbumin. Using overlapping PCR technology twice, firstly two h PTH (1-34) are spliced to obtain the diad, and secondly the diad and H AS are spliced together without any connecting peptide. The fusion protein can replace, delete or add in respective amino acid residue under the premise that the feature of the fusion protein is not changed, prominently extends the half life period in human body based on the condition that the function of the human parathyrine for activating receptor and irritating bone remodeling is retained, and is the medicinal fusion protein which is used for curing osteoporosis and has good perspective in the pharmaceutical field.

Description

technical field [0001] The fusion protein of human parathyroid hormone (1-34) doublet and human serum albumin and its preparation belong to the technical field of long-acting recombinant fusion protein drugs: it involves DNA recombination technologies such as overlapping PCR, and the fusion of drug proteins and HSA makes The drug becomes a long-acting drug. Background technique [0002] Human parathyroid hormone (Parathyroid hormone, PTH) is secreted by human parathyroid chief cells, and it is one of the important hormones in the human body to regulate calcium and phosphorus metabolism. It activates adenylate cyclase and protein kinase C after binding to receptors, changes the rate of bone resorption and bone synthesis, and affects bone metabolism. Studies have shown that intermittent application of PTH in small doses can stimulate bone formation and can effectively treat osteoporosis by inducing bone synthesis. The mature PTH has 84 amino acid residues, but as early as th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/90A61K38/29A61K47/42A61P19/10
Inventor 付强金坚邬敏辰张梅张莲芬李英储敏朱瑞宇陈蕴
Owner JIANGNAN UNIV
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