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Construction, expression and purification method of recombinant human parathyroid hormone in colibacillus

A technology of parathyroid hormone and Escherichia coli, which is applied in the field of medical bioengineering, can solve the problems of high price and high production process cost, and achieve the effect of avoiding renaturation steps and simplifying the purification process

Inactive Publication Date: 2005-12-14
南京大学生物制药工程研究中心
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Problems solved by technology

Since Kex2 protease is expensive and its activity decreases by 50% in the presence of 3M urea, the cost of the production process is relatively high

Method used

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  • Construction, expression and purification method of recombinant human parathyroid hormone in colibacillus
  • Construction, expression and purification method of recombinant human parathyroid hormone in colibacillus
  • Construction, expression and purification method of recombinant human parathyroid hormone in colibacillus

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Embodiment

[0035] 1. Construction of recombinant expression plasmid rhPTH(1-34) / pET-35b(+) Gene construction process ( figure 1 ).

[0036] The rhPTH(1-34) template sequence is: CCGCGG GT TCCGTTTCTGAAATCCAGCTGATGCACAACCTGGGTAAACACCTGAACTCTATGGAACGTGTTGAATGGCTGCGTAAGAAACTGCAGGACGTACACAACTTCTAA CTCGAG (The dashed lines are respectively the restriction sites of SacII and XhoI, and the dashed lines encode the Factor Xa recognition site Ile-Glu-Gly-Arg↓.) The upstream primer sequence is: GGACTG CCGCGG GTATTGAGGGTCGCTCCGTTTC (The underline is the restriction site of SacII.)

[0037] The downstream primer sequence is: GCACAT CTCGAG TTAGAAGTTGTGTACGTCC (the underline is the restriction site of XhoI). The rhPTH(1-34) gene was obtained by PCR amplification, and 0.1 μL of the artificially synthesized rhPTH(1-34) gene template was taken respectively, and the upstream primer and downstream primer were each added to a final concentration of 1 μmol / L, and then 25 mmol / L MgCl was added 2 5 μL...

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Abstract

The present invention relates to medicine biological engineering technology, and is cDNA of small peptide encoding human parathyroid hormone and the method of preparing human parathyroid hormone with its expression vector. Through PCR amplification of chemically synthesized recombinant human human parathyroid hormone (1-34) gene and cloning to expression vector pET-35b(+), recombinant human human parathyroid hormone (1-34) is fused to the carboxyl end of cellulose combining structure domain (CBDclose) and efficient expressed. The fusion protein is treated through cellulose resin affinity chromatographic purification, Factor Xa schizolysis to release rhPTH(1-34), the second cellulose resin affinity chromatography, and C4 reverse efficient liquid chromatographic purification to obtain pure rhPTH(1-34). The sample is tested to have molecular weight of 4117.0 Da.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, including a cDNA encoding human parathyroid hormone small peptide and a method for preparing human parathyroid hormone using its expression vector. Background technique [0002] Parathyroid hormone (PTH) is a linear polypeptide synthesized and secreted by the chief cells of the parathyroid gland [1] . The initial synthesis is preproparathyroid hormone (prepro-PTH) containing 115 amino acids. In the rough endoplasmic reticulum, the N-terminal 25 amino acid residues are removed to form proparathyroid hormone (pro-PTH). Those who remove a hexapeptide from the N-terminal in the Golgi body form a PTH containing 84 amino acids. [0003] PTH is an important hormone that regulates blood calcium levels. Its secretion is mainly regulated by blood calcium concentration. A slight decrease in blood calcium concentration directly stimulates the parathyroid glands to secrete PTH. When blood cal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/16
Inventor 刘建宁孙自勇张菁陈均勇陈新园
Owner 南京大学生物制药工程研究中心
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