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A long-acting recombinant human growth hormone fusion protein and its engineered cells

A technology of human growth hormone and fusion protein, which is applied in the field of genetic engineering and gene recombination of long-acting human growth hormone, can solve the problems of unsatisfactory half-life extension effect of human growth hormone, and achieve the purpose of prolonging serum half-life, improving drug efficacy, improving The effect of stability

Active Publication Date: 2020-05-12
长春生物制品研究所有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the above several studies, the effect of prolonging the half-life of human growth hormone is not satisfactory.

Method used

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  • A long-acting recombinant human growth hormone fusion protein and its engineered cells
  • A long-acting recombinant human growth hormone fusion protein and its engineered cells
  • A long-acting recombinant human growth hormone fusion protein and its engineered cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Design and synthesis of rhGH-Fc fusion protein

[0047] The human antibody IgG4 Fc sequence (DI488112) was mutated at a specific site and then connected to the hGH gene (NM_000515) with a flexible Linker to obtain a mature rhGH-Fc, and the growth hormone signal peptide sequence (CAI94177) was added in front of the hGH sequence to obtain a rhGH-Fc carrying a signal Peptide rhGH-Fc. Whole gene synthesis of mature rhGH-Fc and rhGH-Fc carrying signal peptide, its amino acid sequence is shown in SEQ ID NO: 1; whole gene synthesis of mature rhGH-Fc coding gene, its nucleotide sequence is shown in SEQ ID NO: 2 shown.

[0048] SEQ ID NO 1:

[0049] Amino acid sequence of long-acting recombinant human growth hormone rhGH-Fc immune fusion protein:

[0050] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQ NPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLR SVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFK QTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSV EGSCGFSSASTKGPSVFPLAPGPP...

Embodiment 2

[0056] Example 2: Construction of recombinant expression plasmid containing rhGH-Fc fusion protein coding gene

[0057] (1) Connection of target gene sequence and GS double expression vector

[0058] Take 8.0 μl double expression plasmid p327.8GS respectively (the plasmid map is as follows figure 1 shown) with 0.5μl HindⅢ, 0.5μl EcoRI, 2.0μl Cut Smart buffer, 9.0μl ddH 2 O and 0.5μl XbaⅠ, 0.5μl XhoⅠ, 2.0μl Cut Smart buffer, 9.0μl ddH 2 O The two enzyme digestion systems were mixed in the EP tube, and placed in a 37°C incubator to react for 2 hours for double enzyme digestion. Then 5 μl plasmid GS, 5 μl rhGH-Fc gene sequence carrying signal peptide, 2.0 μl Buffer, 7.0 μl ddH 2 Add O into the EP tube and mix well, add 1.0 μl of melted T4 DNA ligase to the mixed system, mix well, and ligate overnight at 16°C.

[0059] (2) Transformation and extraction of plasmids

[0060] Escherichia coli DH5α and LB medium were inoculated in liquid LB medium (without resistance) at a ratio...

Embodiment 3

[0063] Example 3: CHO-k1 cell culture and transfection

[0064] First, discard all the cell culture medium (preferably IMDM medium) for culturing CHO-k1 cells, add an appropriate amount of HYQtase (if it is an adherent cell), and then put it in CO 2 Perform accelerated digestion at 37°C in an incubator. After the digestion is complete, add an appropriate amount of medium to dilute the digestion solution to terminate the digestion. Pour all the cells into a centrifuge tube, centrifuge at 1500rpm for 5min, and discard the supernatant. Add an appropriate amount of freezing solution (medium + 10% DMSO), mix the cells in the centrifuge tube, count the cells, and make the final concentration 3×10 6 ~1×10 7 Between pieces / ml. The above-mentioned cells were transferred to a pre-cooled cryovial, placed in a freezing box with isopropanol, placed at -80°C overnight and then transferred to liquid nitrogen the next day.

[0065] Remove the frozen plastic tube from the liquid nitrogen t...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to the technical field of gene recombination long-acting human growth hormone. The invention particularly relates to a long-acting recombinant human growth hormone fusion protein and an engineering cell thereof. By the long-acting recombinant human growth hormone fusion protein rhGH-Fc, the serum half-life can be prolonged, the biological activity of the protein in vivo is improved, the immunogenicity of the protein is reduced, therefore, the drug effect is improved, the required injection administration frequency in the treatment time is greatly reduced, the compliance of patients is improved, and convenience of doctors and patients is improved; and the engineering cell for expressing rhGH-Fc constructed by the invention has the characteristics of high protein expression amount and good biological activity, and is particularly suitable for industrial large-scale preparation of gene recombinant long-acting human growth hormone.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the technical field of gene recombinant long-acting human growth hormone, in particular to a long-acting recombinant human growth hormone rhGH-Fc fusion protein, its engineering cells, and its construction and screening methods. Background technique [0002] Human Growth Hormone (hGH) is the most important protein hormone for human growth after birth. It has species specificity and is secreted by eosinophils in the anterior lobe of the human pituitary. The body spreads all over the body, rather than producing physiological effects through the action of target glands. Human growth hormone contains 191 amino acid molecules, most of which exist in the form of a molecular weight of 22KDa, an isoelectric point of 4.9, between the cysteine ​​at the 53rd position and the cysteine ​​at the 165th position and at the 182nd position There are two disulfide bonds between cystein...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10C12N15/66
CPCC07K14/61C07K2319/30C12N15/66C12N15/85
Inventor 刘景会刘涵俞露刘琳琳刘玉林张宇薛毅勇郭胜仓许佳慧
Owner 长春生物制品研究所有限责任公司
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