Production method of recombinant human glandulae parathyroideae (1 to 34 peptide)

A parathyroid hormone, encoding technology, applied in the field of genetic engineering, can solve the problems of low PTH expression, instability, mRNA instability, etc.

Inactive Publication Date: 2008-03-05
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although people use different expression systems to increase the expression of PTH, the expression of PTH is relatively low due to the lack of disulfide bonds in its molecule, its instability, and the instability of mRNA

Method used

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  • Production method of recombinant human glandulae parathyroideae (1 to 34 peptide)
  • Production method of recombinant human glandulae parathyroideae (1 to 34 peptide)
  • Production method of recombinant human glandulae parathyroideae (1 to 34 peptide)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0061] According to the natural amino acid sequence of human parathyroid hormone (1-34 peptide), according to codon preference, under the condition of not changing the amino acid sequence, the target gene of recombinant human parathyroid hormone (1-34 peptide) protein is synthesized completely. Sequence (SEQ ID NO: 1), the homology of the optimized human parathyroid hormone (1-34 peptide) gene sequence and the natural human parathyroid hormone (1-34 peptide) gene sequence is 93% (see Fig. 1). The gene was cloned into pThiHisA and verified by sequencing.

[0062] The expression vector construction method is shown in Figure 2. The target human parathyroid hormone (1-34 peptide) gene was obtained by PCR amplification, and the PCR recovery product of gene human parathyroid hormone (1-34 peptide) and plasmid pThi HisA were respectively processed with BamHI and Ec...

Embodiment 2

[0064] Example 2 Expression of Recombinant Human Parathyroid Hormone (1-34 Peptide) and Determination of Expression Form

[0065] Select a well-confirmed expression engineering strain BL21(DE3) / pThiHisA / PTH, culture it with LB medium and induce expression with IPTG, detect whether there is any target band by SDS-PAGE electrophoresis, and analyze its expression level. Take a part of the fermented bacterial liquid and centrifuge, resuspend in pH 7.4, 20mM PB, + 2.5mM EDTA buffer, ultrasonically destroy the bacteria in an ice bath, collect the ultrasonic supernatant and ultrasonic precipitation by centrifugation, detect by SDS-PAGE electrophoresis, and determine its ways of expression. Analysis of the ultrasonic supernatant and ultrasonic precipitation revealed that the target protein was mostly in the precipitate, indicating that the target protein was expressed in a soluble form (see Figure 3).

Embodiment 3

[0066] Example 3 Scale Fermentation of Recombinant Human Parathyroid Hormone (1-34 Peptides)

[0067] Take the expression engineered bacteria BL21(DE3) / pThiHisA / PTH identified as a positive clone, and culture it in LB containing 50 μg / mL ampicillin for 14-16 hours; 600 1 to 3, as the secondary seed liquid. Prepare 2.96L of improved M9 fermentation medium (Glucose1%, Yeast extract0.5%, K 2 HPO 4 0.5%, KH 2 PO 4 0.35%, (NH 4 ) 2 HPO 4 0.35%, MgSO 4 0.025%, CaCl 20.1%), pour it into a cleaned fermenter, after autoclaving the tank, cool to 30-40°C, put the cultured secondary seed liquid into the fermenter at 1.5% inoculum, cultivate at 37°C, and Constantly adjust the ventilation flow rate and stirring speed according to the dissolved oxygen value, and control the dissolved oxygen above 40%. Sampling every hour to measure OD 600 value. Grow to OD 600 When it reaches about 5.0, the induction starts. Add IPTG to the final concentration of 1mM in the fermenter for inducti...

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Abstract

The present invention provides one nucleotide sequence of human parathyroid hormone (1-34 peptides), the process for producing recombinant human parathyroid hormone (1-34 peptides) in high efficiency, and relevant engineering cell constituting, expressing and purifying process. The purified recombinant human parathyroid hormone (1-34 peptides) protein gene is especially suitable for expression in prokaryotic cell, and has the advantages of high expression quantity and high stability after being optimized through a fermenting and purifying process. The present invention can obtain pure recombinant human parathyroid hormone (1-34 peptides) product simply in high efficiency and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a method for efficiently producing recombinant human parathyroid hormone (1-34 peptides), including the construction of related engineering bacteria, the expression of recombinant human parathyroid hormone (1-34 peptides) and Purification process. Background technique [0002] In 1925, Collip isolated parathyroid hormone from bovine parathyroid gland tissue, but the extracted product was unstable and the active components were not single. After that, Aurbach (1959) obtained a uniform and stable product by phenol extraction until 1974 Henry et al. Only by using phenol, trichloroacetic acid and column chromatography can the product with high purity be obtained, and it can be used in the research of biological function and chemical structure. [0003] Human parathyroid hormone (human parathyroid hormone, PTH) is a straight-chain polypeptide hormo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/16C07K14/635C12N15/63C12P21/02C12N1/21
Inventor 黄阳滨孙九如张翊任军梁光军邱燕
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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