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Recombinant human growth hormone, and preparation method and drug applications thereof

A technology for human growth hormone and seeds, applied in the fields of protein purification and genetic engineering, can solve the problems of low secretion efficiency, limited periplasmic space of Escherichia coli, low production of secreted rhGH, etc., and achieves clear medium components and good physiological activity. , effects that are easy to zoom in and repeat

Pending Publication Date: 2020-06-19
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the periplasmic space of Escherichia coli is relatively limited, and the secretion efficiency is generally not high, so the production of secreted rhGH is generally not high

Method used

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  • Recombinant human growth hormone, and preparation method and drug applications thereof
  • Recombinant human growth hormone, and preparation method and drug applications thereof
  • Recombinant human growth hormone, and preparation method and drug applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of rhGH Escherichia coli strain

[0042] The inventors directly fused the OmpA3 signal peptide and the rhGH gene (GenBank accession number: NM_000515.4), optimized the codons for Escherichia coli, optimized the mRNA free energy, and combined the optimized OmpA3 gene (as shown in SEQ ID NO: 2 ), rhGH gene (as shown in SEQ ID NO: 1) was combined and entrusted to GenScript Biotechnology Co., Ltd. to synthesize (denoted as OmpA3-rhGH), and the synthesized gene was inserted into the pUC57 vector to construct a recombinant cloning plasmid, denoted It is pUC57-OmpA3-rhGH; the plasmid pUC57-OmpA3-rhGH was introduced into the expression cloning host of Escherichia coli Top10 (referred to as Top10-pUC57-OmpA3-rhGH) by means of molecular cloning. To clone the rhGH gene from the pUC57 vector into pDEST14, see the following steps:

[0043] 1. Take glycerol tubes of Top10-pUC57-OmpA3-rhGH and DH5α-pDEST14 strains (preserved in our laboratory) frozen in a -80°C...

Embodiment 2

[0051] Embodiment 2 rhGH small test fermentation (20L)

[0052] This example mainly describes the 30L fermentation process of BL21AI-pDEST14-OmpA3-rhGH bacterial strain. In the fermentation process, glycerol with clear composition is used as the carbon source for cell metabolism, and ammonia water is used (for both pH adjustment and Nitrogen source is used for cell metabolism) and diammonium hydrogen phosphate as the nitrogen source for cell metabolism; at the same time, in order to promote cell growth, shorten the fermentation cycle, and increase fermentation yield, compound nutrients such as yeast powder and peptone have also been added to the medium. This embodiment mainly introduces the fermentation process conditions of rhGH 20L scale, and the fermentor used is a Sartouris cplus fermenter, and the initial volume of fermentation is 20L.

preparation example 1

[0053] Preparation example 1 concrete steps are as follows:

[0054] 1. Preparation of fermented seed liquid: Inoculate the rhGH Escherichia coli strain cryopreservation tube constructed in Example 1 into LB solid medium containing 100 μg / ml ampicillin using the three-section method, and culture overnight at 37°C activation.

[0055] 2. Pick a single colony with a plump shape and a moderate size from the solid medium and inoculate it into LB liquid medium containing 100 μg / ml ampicillin, and culture it on a shaking table at 37°C and 220 rpm for 8 hours. This is the primary seed solution.

[0056] 3. Transfer the primary seed solution in step 2 to fresh LB liquid medium containing 100 μg / ml ampicillin according to the inoculum amount of 1%, and cultivate it on a shaker at 37°C and 220 rpm until OD600≈3-5 , which is the preparation of secondary seed solution.

[0057] 4. Composition of batch fermentation medium: citric acid monohydrate 1.5g / L, potassium dihydrogen phosphate 13...

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Abstract

The invention provides a recombinant human growth hormone, and a preparation method and drug applications thereof. The optimization of the gene of rhGH suitable for the expression of an escherichia coli system, the construction of suitable expression vectors and strains, the development of rhGH fermentation technologies are specifically related, and the development of rhGH purification technologies, especially the optimization of extraction technologies of periplasmic proteins are specifically related. A provided rhGH secretory expression system can effectively realize the secretion of the rhGH and the thorough excision of signal peptide. The produced rhGH has good physiological activity; the technologies have the characteristics of being clear in culture medium components, low in cost andeasy to amplify and repeat; and an provided extraction method of the periplasmic proteins is good in repeatability, high in extraction efficiency and good in selectivity. The purity, endotoxin, HCP and DNA residues of purified samples can meet medicinal standards.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and protein purification, and in particular relates to a recombinant human growth hormone and its preparation method and pharmaceutical application. Background technique [0002] Human Growth Hormone (hGH) is a protein hormone with a single peptide chain secreted by eosinophilic cells in the anterior pituitary gland. It is the most important hormone that promotes growth after birth. It has multiple functions such as regulating human growth and metabolism. . It contains 191 amino acid residues, the molecular weight is about 22k Da, and there is no glycosylation in the molecule. Human growth hormone has a wide range of physiological functions and can affect almost all tissue types and cells, even including immune tissue, brain tissue and hematopoietic system. [0003] Human growth hormone (hGH) plays an important role in the growth and development of the human body. It can promote the growth of...

Claims

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Application Information

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IPC IPC(8): C07K14/61C07K1/36C07K1/34C07K1/30C07K1/16C07K1/14C12N15/70C12N1/21C12P21/02C12R1/19
CPCC07K14/61C12N15/70
Inventor 马永赵百学王安良
Owner ZONHON BIOPHARMA INST
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