Method for producing recombined human parathyroid hormone (1í½84peptide)

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

Inactive Publication Date: 2008-04-02
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although people use different expression systems to increase the expression of PTH, the expression of PTH

Method used

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  • Method for producing recombined human parathyroid hormone (1í½84peptide)
  • Method for producing recombined human parathyroid hormone (1í½84peptide)
  • Method for producing recombined human parathyroid hormone (1í½84peptide)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0057] According to the natural amino acid sequence of human parathyroid hormone (1-84 peptide), according to codon preference, under the condition of not changing the amino acid sequence, the target gene of recombinant human parathyroid hormone (1-84 peptide) protein was synthesized by PCR method Sequence (SEQ' ID NO: 1), the homology between the optimized IL-4 gene sequence and the natural IL-4 gene sequence is 82% (see Figure 1). The gene was cloned into pThiHisA and verified by sequencing.

[0058] The expression vector construction method is shown in Figure 2. The target human parathyroid hormone (peptide 1-84) gene was amplified by PCR, and the PCR recovery product of the gene PTH (peptide 1-84) and plasmid pThiHisA were respectively treated with BamHI and EcoRI double digestion, and the corresponding pThiHisA was recovered after digestion. Fragments w...

Embodiment 2

[0060] Example 2 Expression of Recombinant Human Parathyroid Hormone (1-84 Peptide) and Determination of Expression Form

[0061] 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 break the bacteria in an ice bath, collect the ultrasonic supernatant and ultrasonic precipitation by centrifugation, detect by SDS-PAGE electrophoresis, and determine its expression form. 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

[0062] Example 3 Scale Fermentation of Recombinant Human Parathyroid Hormone (1-84 Peptides)

[0063] 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 (Glucose 1%, Yeast extract 0.5%, K 2 HPO 4 0.5%, KH 2 PO 4 0.35%, (NH 4 ) 2 HPO 4 0.35%, MgSO 4 0.025%, CaCl 2 0.1%), 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 ...

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Abstract

The present invention provides a nucleotide sequence of recombinant human parathyroid hormone (1 to 84 peptides) as well as a high efficiency production method of the recombinant human parathyroid hormone (1 to 84 peptides), and construction, expression and purification art of related engineered cells. The protein gene of the optimized recombinant human parathyroid hormone is very suitable for expressing in yeast and improves the yield by optimizing with ferment and purification art at the same time, and has the advantages of high expression and high stability. The present invention can obtain highly efficiently and conveniently the pure recombinant human parathyroid hormone at lower 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 (1-84 peptides), including optimization of related genes, construction of engineering bacteria, recombinant human parathyroid hormone (1-84 peptides) ) expression and purification process. Background technique [0002] The basic physiological function of PTH is to stimulate bone resorption by osteoclasts, release calcium and phosphorus from bones, promote calcium reabsorption by renal tubules, inhibit phosphorus reabsorption, and increase the conversion of 25-(OH)D3 into 1,25- (OH)2D3 promotes the reabsorption of calcium and phosphorus in the intestine. Studies have shown that PTH can directly increase the number of osteoclasts, increase the folded edge of osteoclasts, increase the activity of osteoclasts, and promote bone resorption. However, PTH does not directly act o...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/16C07K14/635C12N15/63C12N1/21C12P21/02
Inventor 黄阳滨孙九如张翊任军张继岗梁光军邱燕
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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