Prepn. of recombinant human parathyroid hormone PTH(1-34)

A parathyroid hormone, crude technology, applied in the direction of parathyroid hormone, hormone peptide, recombinant DNA technology, etc., can solve the problems of high cost, unfavorable large-scale production, expensive enterokinase, etc.

Inactive Publication Date: 2005-07-27
西南生物工程产业化中试基地有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, enterokinase is expensive, making the process costly and unfavorable for large-scale production

Method used

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  • Prepn. of recombinant human parathyroid hormone PTH(1-34)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the construction of engineering bacteria

[0042] 1) Select pThio-HisA as the expression vector,

[0043] 2) Cut out the gene fragment from the PUC-18 plasmid with EcoR I-Sal I, separate it with 1% Agarose gel, and purify it for future use. Digestion conditions: 10*Buffer: 5 μl, DNA: 30 μl, EcoR I 1 μl, Sal I 1 μl, water 13 μl, 37°C water bath for 2 hours.

[0044] 3) The pThio His A vector was digested with EcoR I-Sal I, treated with CAP, and purified for later use. Digestion conditions: 10*Buffer: 5 μl, DNA: 30 μl, EcoR I 1 μl, Sal I 1 μl, water 13 μl, 37°C water bath for 2 hours.

[0045] 4) Cloning the small and medium fragments in step 2) to the large fragment obtained in step 3), mixing the carrier fragment and the target fragment at a ratio of 1:3, and using T 4 The ligase was ligated overnight at 16°C to construct a recombinant plasmid.

[0046] 5) Transform Escherichia coli BL21 competent cells with the recombined plasmid, spread it on an LB p...

Embodiment 2

[0050] Embodiment 2: Purification of PTH

[0051] According to the method of the present invention, PTH expressing bacteria are constructed, cultured by fermentation, and the bacteria are collected by centrifugation. The bacteria were suspended in lysate (50 mM Tris, 150 mM NaCl, pH 8.0), ultrasonically disrupted, and the inclusion bodies were collected by dilution and centrifugation. And washed with lysate, and centrifuged to further purify the PTH inclusion body.

[0052] 20 g of inclusion bodies were suspended in 50 ml of 50 mM Tris, 150 mM NaCl (pH 8.0), 36 g of solid urea was slowly added under electromagnetic stirring to a final concentration of 6 M, and stirring was continued for 4 hours. The insoluble matter was removed by centrifugation, and 450 ml of the supernatant was diluted with 50 mM Tris, 150 mM NaCl (pH 8.0) until the concentration of urea was reduced to 1 M for refolding overnight. The solution after renaturation was centrifuged to remove impurity proteins,...

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Abstract

The present invention relates to a preparation process of recombinant human parathyroid hormone PTH(1-34). The process constitutes a new engineering strain to result in high expression amount, simple purifying course and low cost.The present invention adopts colibacillus degenerate codon modifying sequence corresponding cDNA, the basic sequence is L-V-P-R-PTH(1-34), where L-V-P-R is the enzyme incision site of thrombin. To the end of the nucleotides sequence, colibacillus terminator codon TAA is added; and in order to clone easily to the 5'-terminal enzyme incision site of EcoR I is added, and to the 3'-terminal, enzyme incision site of Sal I is added. High-purity recombinant huamn parathyroid hormone PTH(1-34) is obtained through fermentation of the engineering bacteria, occlusion body extraction, diluting renaturation, thrombase incision, anionic exchange chromatography and other steps.

Description

Technical field: [0001] The invention relates to a preparation method of human parathyroid hormone PTH (1-34), especially a method for preparing the product by using gene recombination technology, and the product can be used for treating osteoporosis. Background technique: [0002] Osteoporosis is a type of disease related to bone metabolism, which is a non-specific bone metabolic disorder, mainly occurring in the spongy bone part of the bone. Bone metabolism includes two aspects: osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Bone metabolism is a dynamic balance process between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. [0003] With the increase of age, bone resorption activity exceeds bone formation activity, resulting in bone loss, bone density gradually decreases, and the bones are hollow and loose, fragile and easy to fracture, which is called osteoporosis. Patients with osteoporosis are prone to fractures...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/635C12N1/21C12N15/63C12N15/64C12P21/02
Inventor 胡昌华李明何建军徐辉
Owner 西南生物工程产业化中试基地有限公司
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