Human auxin fusion protein TAT-hGH as well as preparation method and application thereof

A technology of fusion protein and human growth hormone, applied in the field of strain E.coli Orig, can solve the problems of inconvenient treatment of patients, cumbersome methods, and difficulty in popularizing anti-aging use, and achieve the effects of increasing utilization efficiency, reducing pain, and improving drug efficacy.

Active Publication Date: 2013-03-27
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] As a protein hormone that can stimulate growth, hGH has been widely used clinically to treat diseases such as growth hormone deficiency, extensive burns, and short bowel syndrome. Daniel Rudman published his paper in the "New England Journal of Medicine" that shocked the medical community - Effects of human growth hormone in men over 60 years old. The role of human growth hormo

Method used

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  • Human auxin fusion protein TAT-hGH as well as preparation method and application thereof
  • Human auxin fusion protein TAT-hGH as well as preparation method and application thereof
  • Human auxin fusion protein TAT-hGH as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Construction and identification of embodiment 1 recombinant plasmid pMD-TAT-hGH

[0057] 1.PCR

[0058] Primers were designed according to the sequence of hGH (GenBank: V00520.1) and the base sequence of 11 amino acids in TAT PTD. The primers were designed as follows:

[0059] Upstream primer 1: 5`-AAACGTCGTCAGCGTCGTCGTTTCCCAACCATTCCC-3`

[0060] Upstream primer 2: 5`-GAATCC CATATG TATGGCCGTAAGAA-3` (the Nde I restriction site is underlined)

[0061] Downstream primer 1: 5`-TTA CTCGAG GAAGCCACAGCTGCCCT-3` (the underline is the XhoI restriction site)

[0062] The above primers were synthesized by Yingjun Company.

[0063] Synthesize TAT-hGH by PCR in 3 times: use pMD18T-hGH plasmid (synthesized by Generay) as template, upstream primer 1, downstream primer 1, annealing temperature 55°C, perform PCR amplification of the target fragment hGH, electrophoresis and recover; the above step The recovered product was used as a template, the upstream primer 2, the downstrea...

Embodiment 2

[0135] Example 2 Construction of genetic engineering strain E.coli Origami B(DE3)pLysS(pET-TAT-hGH)

[0136] 1. Double digestion

[0137] The pET-22b(+) plasmid (purchased from Novagen) and the pMD-TAT-hGH plasmid DNA were subjected to double digestion. The reaction system is shown in the table below:

[0138]

[0139] Mix and mix the above reaction system components on ice, centrifuge briefly at 5000rpm, place in a 37°C water bath for 3 hours, add 0.1 times the volume of 10×loading buffer to terminate the reaction, all used in 1% agar Sugar gel electrophoresis. TAT-hGH and pET-22b(+) fragments were recovered.

[0140] 2. Connect

[0141] The TAT-hGH recovered in 1 and the pET-22b(+) fragment were mixed on ice and added as shown in the table below.

[0142]

[0143]

[0144] Put 10 μl of the mixed solution in a sterile PCR tube, use a pipette gun to gently suck a few times to mix, centrifuge briefly at 5000 rpm, concentrate the mixed solution at the bottom of the...

Embodiment 3

[0164] The expression of embodiment 3 genetic engineering fusion protein TAT-hGH

[0165] Pick a single colony transformed with the plasmid, inoculate it in 5ml of selective LB liquid medium, and cultivate overnight at 37°C with shaking at 250rpm / min. On the next day, re-inoculate 200 μl of overnight cultured bacteria into 20ml (1:100) selective 2YT liquid medium (17g peptone, 10g yeast extract, 5g NaCl, add water to 1000ml), shake at 250rpm / min at 37°C Cultured to optical density (OD 600 =0.6), add 1mol / L IPTG to the bacterial solution to make the final concentration of IPTG 1mM, culture at 37°C and shake at 250rpm / min for 4 hours.

[0166] Take 1ml of the fermentation broth in a 1.5ml EP tube, centrifuge at 13000rpm for 1min, suck off the supernatant, resuspend the precipitate with deionized water, and use it as the cell sample solution for SDS-PAGE gel. Take another 4ml of fermentation broth in a 1.5ml EP tube, centrifuge at 13000rpm for 1min, collect the precipitate, add...

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Abstract

The invention discloses fusion protein TAT-hGH of human auxin and cell-penetrating peptide TAT as well as a preparation method and application of the fusion protein TAT-hGH. A deoxyribonucleic acid (DNA) sequence of the fusion protein TAT-hGH, a carrier containing the DNA sequence and a host cell containing the carrier are encoded; and a method for preparing the fusion protein TAT-hGH by gene engineering is provided. The fusion protein TAT-hGH has the functions of promoting Jurkat cell proliferation activity and influencing a transcriptional level of insulin-like growth factor (IGF) I of downstream factors; and meanwhile, the fusion protein TAT-hGH has a function of penetrating through intestines. The fusion protein TAT-hGH can be orally taken, enters blood by penetrating through an intestinal cell membrane, and can reach different tissues by blood circulation, so that the utilization ratio of hGH is increased, and the medicinal effect is also improved. The preparation method can be applied to large-scale industrial production, and is high in expression amount, easy to operate and low in cost.

Description

technical field [0001] The invention relates to the technical field of genetic bioengineering, in particular to a human auxin TAT-hGH recombinant fusion protein, and more specifically to a genetic engineering strain E.coli Origami B(DE3)pLysS(pET-TAT-hGH containing human auxin gene ), the present invention also relates to the preparation method of the TAT-hGH recombinant fusion protein and the application of the fusion protein TAT-hGH in promoting cell proliferation, promoting animal growth and penetrating intestines. Background technique [0002] Human growth hormone (hGH) is a protein hormone synthesized and secreted by the anterior lobe of the human pituitary gland. Growth hormone is first translated into prohormone, that is, growth hormone precursor protein. The growth hormone precursor protein contains 217 amino acids, and the 1-26 amino acids at the amino terminal are signal peptides. If the signal peptide cannot be specifically excised, the hormone cannot produce acti...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12P21/02A61K38/27A61K47/48A61P5/06C12R1/19A61K47/42
Inventor 徐进平孟小林王健倪雅雯
Owner WUHAN UNIV
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