Recombinant human growth hormone and its encoding gene, preparation method and application

A technology of human growth hormone and encoding gene, which is applied to the preparation method of growth hormone, peptide, hormone peptide, etc., can solve the problems of limited source and high price, and achieve the effects of simple steps, low cost and high protein expression.

Active Publication Date: 2021-01-26
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the past, the clinical application could only be extracted from the human pituitary gland, the source was very limited, and the price was very expensive.

Method used

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  • Recombinant human growth hormone and its encoding gene, preparation method and application
  • Recombinant human growth hormone and its encoding gene, preparation method and application
  • Recombinant human growth hormone and its encoding gene, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Construction and expression identification of rhGH Escherichia coli strain

[0046] The inventor optimized the hGH gene (GenBank accession number: NM_000515.4) for E. coli codons and mRNA free energy, and entrusted GenScript Biotechnology Co., Ltd. to synthesize the optimized rhGH gene (SEQ NO.1). The synthesized gene was inserted into the pUC57 vector to construct a recombinant cloning plasmid, denoted as pUC57-rhGH; the plasmid pUC57-rhGH was introduced into the expression cloning host of E. coli Top10 (denoted as Top10-pUC57-rhGH) by means of molecular cloning . To clone the rhGH gene from the pUC57 vector into pET28a, see the following steps:

[0047] 1. Take glycerol tubes of Top10-pUC57-rhGH and DH5α-pET28a strains (purchased from Nanjing GenScript) frozen in a -80°C refrigerator, and inoculate the former in LB liquid medium containing 100 μg / ml ampicillin after thawing. The latter was inoculated in LB liquid medium containing 50 μg / ml kanamycin, cultu...

Embodiment 2

[0054] Embodiment 2 rhGH high-density fermentation

[0055] This example mainly describes the high-density fermentation process of the BL21(DE3)-pET28a-rhGH bacterial strain. In the fermentation process, glucose and glycerol with definite components are used as the carbon source for cell metabolism, and ammonia water (used for pH adjustment during the fermentation process) Also used as a nitrogen source for cell metabolism) and diammonium hydrogen phosphate as a nitrogen source for cell metabolism; due to avoiding the use of compound media similar to yeast powder and peptone, it avoids the compound nitrogen source / carbon source. Therefore, the medium composition of different fermentation batches can be easily controlled, and because the cost of glucose and glycerol is low, the overall cost of the fermentation process is well controlled. Fermentation cell OD after fermentation 600 It can reach between 90-120, and the expression level of the target protein can reach more than 1...

preparation example 1

[0056] Preparation example 1 concrete steps are as follows:

[0057] 1. Preparation of fermented seed liquid: inoculate the rhGH Escherichia coli strain cryopreservation tube constructed in Example 1 into LB solid medium containing 50 μg / ml kanamycin using the method of three-section line, and overnight at 37°C culture for activation;

[0058] 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 50 μg / ml kanamycin, and culture it on a shaker at 220 rpm at 37°C for 8 hours. This is the primary seed solution.

[0059] 3. Transfer the primary seed liquid in step 2 to fresh LB liquid medium containing 50 μg / ml kanamycin according to the inoculum amount of 1%, and cultivate it on a shaker at 37°C and 220 rpm until OD600≈3-5 Between, this is the preparation of secondary seed solution.

[0060] 4. Composition of batch fermentation medium: citric acid monohydrate 1.5g / L, potassium dihydrogen phosphate ...

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Abstract

The invention relates to a recombinant human growth hormone and its encoding gene, preparation method and application, belonging to the field of genetic engineering. The invention provides a preparation method of Escherichia coli expression, inclusion body renaturation and purification of recombinant human growth hormone (rhGH). Specifically involved: 1. Construction of rhGH Escherichia coli expression strain; 2. Development of rhGH high-density fermentation process; 3. Development of rhGH inclusion body denaturation and renaturation process; 4. Purification and preparation of rhGH samples after renaturation. Through the above four levels of technical research, the rhGH produced by the patented method has high biological activity, and the method has high yield, low cost, and is easy to scale up. It can be used as a drug for treating diseases caused by lack of growth hormone.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a method for recombinant human growth hormone (rhGH) and its coding gene, expression in Escherichia coli, renaturation and purification of inclusion bodies, and its preparation and treatment of diseases caused by lack of growth hormone application in medicines. 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 syst...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/61C07K1/34C12N15/70C12P21/02A61K38/27A61P39/06A61P19/10A61P9/00
CPCA61K38/00C07K14/61C12P21/02
Inventor 马永赵百学王安良江辰阳吴诚
Owner ZONHON BIOPHARMA INST
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