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

A parathyroid hormone and expression vector technology, applied in the field of genetic engineering, can solve the problems of low PTH expression, unstable mRNA, 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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  • Cascade expression method of recombinant human glandulae parathyroideae (1 to 34 peptide)
  • Cascade expression method of recombinant human glandulae parathyroideae (1 to 34 peptide)
  • Cascade expression method of recombinant human glandulae parathyroideae (1 to 34 peptide)

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Experimental program
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Effect test

Embodiment 1

[0061] Example 1 Construction of expression plasmids and acquisition of high-expression engineering strains

[0062] 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 from the whole gene Sequence (SEQ ID NO: 1), the homology between the optimized PTH gene sequence and the natural PTH gene sequence is 85% (see Figure 1). The gene was cloned into pThiHisA and verified by sequencing.

[0063] The expression vector construction method is shown in Figure 2. Introduce a Lys(K), Arg(R) and BglII restriction site at the front 5' end of each monomeric gene, and introduce a Lys(K), Arg(R) and BamHI restriction site at its 3' end . The target human parathyroid hormone (1-34 peptide) gene was amplified by PCR, and the PCR product was double digested with BglII an...

Embodiment 2

[0066]Select a well-confirmed expression engineering strain BL21(DE3) / pThiHisA / PTH-fusion, 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 showed that most of the target protein was in the ultrasonic supernatant, indicating that the target protein was expressed in a soluble form.

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Abstract

The present invention provides one nucleotide sequence of human parathyroid hormone, the serial expression process for producing recombinant human parathyroid hormone in high efficiency, and relevant engineering cell constituting, expressing and purifying process. The purified recombinant human parathyroid hormone protein gene is especially suitable for serial 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 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) peptide, including the acquisition of related concatemers, construction of engineering bacteria, recombinant human parathyroid hormone (1-34 ) expression of the peptide concatemer and the corresponding 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 parathy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/16C07K14/635C12N15/66C12P21/02C12N1/21
Inventor 黄阳滨孙九如张翊任军梁光军邱燕
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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