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Recombinant human growth hormone Fc fusion protein, application and engineering cell strain of fusion protein

A human growth hormone and fusion protein technology, which is applied in the field of the development and use of recombinant human growth hormone Fc fusion protein and its engineering cell lines, can solve the problems of low affinity and achieve the effect of long half-life

Pending Publication Date: 2021-04-16
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the four human IgG subtypes, IgG1 and IgG3 can effectively bind to the Fc receptors on the cell surface, while IgG2 and IgG4 bind to the receptors with lower affinity

Method used

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  • Recombinant human growth hormone Fc fusion protein, application and engineering cell strain of fusion protein
  • Recombinant human growth hormone Fc fusion protein, application and engineering cell strain of fusion protein
  • Recombinant human growth hormone Fc fusion protein, application and engineering cell strain of fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Construction of the expression vector of rGH-Fc / Fc-rGH

[0032] 1.1 Synthetic GH gene

[0033] Retrieve the amino acid sequence of GH from the NCBI database (GenBank Accession: M13438.1), analyze its nucleotide sequence, carry out modifications such as de-correlating enzyme cleavage sites or reducing the stem-loop structure for the nucleotide sequence, and entrust the whole gene synthesis of rGH-Fc For the full-length gene, its nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2.

[0034] 1.2PCR amplification of XbaI-rGH-Fc-HindIII and XbaI-Fc-rGH-XhoI

[0035] Design the following primers:

[0036]

[0037]

[0038] GH-Fc amplification: using the synthetic GH gene-containing plasmid (pUC57-GH) as a template, using P1 and P2 as primers, and using Prime STAR HS polymerase, PCR amplified to obtain the XbaI-GH fragment. Using the existing IgG-Fc gene plasmid as a template, using P3 and P4 as primers, and u...

Embodiment 2

[0051] Embodiment 2: rGH-Fc / Fc-rGH protein expression and purification

[0052] 2.1 Expression and identification of rGH-Fc / Fc-rGH

[0053] Inoculate Expi293 cells into 24-well culture plates at a seeding density of 2.5-3×10 6 cells / ml, the inoculum volume is 1ml / well, and the cell viability is greater than 90% during transfection. Dilute 1 μg DNA (pCD-rGH-Fc / Fc-rGH) in 50 μl OptiMEM, mix gently, then dilute 2.7 μl in 50 μl OptiMEM, mix gently, incubate at room temperature for 5 minutes, and mix the obtained DNA dilution and the obtained Mix ExpiFectamine dilutions, mix gently, incubate at room temperature for 20 minutes, add the resulting DNA-ExpiFectamine complex (total volume about 100 μl) into the culture well plate, shake the culture plate back and forth to make it evenly distributed. Put the cells in the incubator and continue to culture for 16 hours, add transfection Enhancer1 at the ratio of 5 μl / ml, add transfection Enhancer2 at the ratio of 50 μl / ml, add antibiotic...

Embodiment 3

[0056] Example 3: Binding of rGH-Fc / Fc-rGH protein to GHR

[0057] Growth hormone receptor protein (GHR) was diluted to 1 μg / ml with PBS, and plated overnight at 4°C. Wash 3 times with TPBS, block in an oven at 37°C for 1 hour, wash 3 times with TPBS, add gradiently diluted rGH-Fc / Fc-rGH fusion protein (50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19nM), 100 μl per well and incubated for 1 hour at room temperature with shaking. After washing 3 times with TPBS, add sheep anti-human Fc-HRP (1:4000 dilution), 100 μl per well, incubate with shaking at room temperature for 1 hour, wash 3 times with TPBS, add OPD for color development, react at room temperature for 10 minutes, and then add 1M H2SO4 to stop the reaction. Wells 100 μl, absorbance was measured at 490 nm. Test results such as Figure 4 As shown, the results showed that both rGH-Fc and Fc-rGH could bind to GHR, and showed a concentration-gradient-dependent trend.

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Abstract

The invention relates to the technical field of biomedical engineering, in particular to the technical field of gene recombination long-acting human growth hormone, and particularly relates to development of recombination human growth hormone Fc fusion protein. The human growth hormone Fc fusion protein of the present invention contains human growth hormone (hGH), a flexible peptide linker (L) and a human immunoglobulin Fc fragment. The invention also discloses an activity determination method of the fusion protein and a host cell capable of expressing the fusion protein. Compared with recombinant hGH, the recombinant human growth hormone Fc fusion protein constructed by the invention has a longer serum half-life period, and the injection administration frequency required in the treatment time is greatly reduced, so that the compliance of patients is improved, the convenience of doctors and patients is improved, the production process is simpler and more efficient, and the fusion protein is especially suitable for industrial large-scale preparation of the gene recombinant long-acting human growth hormone.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to the technical field of gene recombinant long-acting human growth hormone, in particular to the development and use of a recombinant human growth hormone Fc fusion protein and its engineering cell strain. Background technique [0002] Growth hormone (HGH) is a peptide hormone that is synthesized, stored and secreted by somatotroph cells in the pituitary gland. Mature wild-type human GH (hGH identified by SEQ ID NO: 2) is composed of 191 amino acid residues, most of which exist in the form of molecular weight 22KDa, a small amount of growth hormone exists in the form of 20KDa, and the isoelectric point is 4.9. Positions 53, 165, 182 and 189 contain four cysteine ​​residues, which stabilize the three-dimensional structure of the protein by forming two intramolecular disulfide bonds connecting C53 and C165 and C182 and C189, respectively. Growth hormone and its specif...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/72C12N15/62C12N15/85C12N5/10C12P21/02A61K38/27A61P5/06A61P17/02C12R1/91
Inventor 徐婷杨文静许文娟黄伟叶洪涛崔智强范清林宋礼华
Owner ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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