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Recombinant human nerve growth factor deletion mutant, its preparation method and application

A technology for deletion of mutants and growth factors, applied in the field of mutants, can solve the problems of rhNGF quality control and other problems, and achieve the effects of maintaining the biological activity, the C-terminal of the protein is uniform, and the protein expression level is improved.

Active Publication Date: 2015-04-29
军事科学院军事医学研究院生物工程研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using eukaryotic cells to prepare rhNGF, it is also found that there are often 2 to 3 amino acid deletions at the C-terminal, which will form homologous or heterodimers, such as 120 / 120, 118 / 118, 117 / 117, 120 / 118, 117 / 118, etc., which caused some troubles to the quality control of rhNGF

Method used

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  • Recombinant human nerve growth factor deletion mutant, its preparation method and application
  • Recombinant human nerve growth factor deletion mutant, its preparation method and application
  • Recombinant human nerve growth factor deletion mutant, its preparation method and application

Examples

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Embodiment 1

[0032] Example 1 Recombinant human nerve growth factor deletion mutant (rhNCF-D3) gene cloning and vector construction

[0033] Design primers according to the published human NGF DNA sequence (Genbank: NM_002506), isolate human peripheral blood albumin, use TriZol (purchased from Invitrogen), extract its total RNA, reverse transcribe into cDNA, reverse transcription system is template RNA 2 μg , 5× reaction solution 4μl, dNTP mixture (10mM, each) 1μl, RNase inhibitor (20U / μl), random primer 2μl, M-MLV reverse transcriptase (200U / μl) 1μl, add water to make up the total volume to 20μl. After standing at room temperature for 10 min, react at 42°C for 1 h. Using this cDNA as a template, the human NGF gene and the deletion mutant gene were amplified. The upstream primers for amplification were "5'-atgaa ttcca ccatg tccat gttgt tctac actc t ga-3'", and the downstream primers were "5'-atccc gggtt atcag gctct tctca cagcc ttcct gct-3' (for full-length NGF gene amplification)" and "5'...

Embodiment 2

[0038]The prepared pCI-neo NGF and pCI-neo NGF-D3 plasmids were electrotransformed into CHO S cells (purchased from Invitrogen Company), the electroporation apparatus was Gene pulser Xcell (BIO-RAD Company), and the electroporation conditions were 160V, 150ms. The medium is DMEM with 10% fetal bovine serum added. After electroporation, the cells in the electroporation cup are washed out with the medium, and spread in a 35mm cell culture dish. On the third day, 600 μg / ml 6418 (Sigma company) was added to the culture medium to pressurize and select resistant cells, and the surviving monoclonal cell clusters after resistance selection were transferred to a 96-well plate, and the recombination in the cell supernatant was detected by dot-blot Protein expression level, select high-expressing cell clones into suspension culture.

[0039] The host cells are preferably mammalian cells, and Chinese hamster ovary (CHO) cells are used in this example, and human embryonic kidney 293 cells,...

Embodiment 3

[0041] Select cell lines with better expression levels and transfer them to 40ml shake flasks (Corning Company), select cell lines adapted to serum-free suspension culture, the medium used is SFM4 (Hyclone Company), and the culture conditions are 37°C, compare the cell growth curves , Recombinant protein expression level (quantitative ELISA detection, BD company, dy265), and finally determined the recombinant cell line 1F1G8 (expressing full-length recombinant human nerve growth factor (rhNGF)) and cell line 2F5 (expressing recombinant human nerve growth factor C-terminal deletion 3 amino acid mutant (rhNGF-D3)) as a recombinant cell line for further research.

[0042] The cell lines 1F1G8 and 2F5 were explored in a small-scale batch fed-batch cell culture process in a 10L WAVE bioreactor (Satorious Company), and the two cell lines maintained similar growth curves, such as Figure 4 As shown, but the expression level of rhNGF-D3 protein is about 10 times higher than that of rh...

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Abstract

Disclosed is a deletion mutant of a recombinant human nerve growth factor. The mutant is a peptide chain of the complete human nerve growth factor, with 3 amino-acids at C-terminal deleted, wherein the amino-acid sequence of the mutant is as shown in sequence list SEQ ID No.1, or the amino-acid sequence of the mutant is an amino-acid sequence that shares over 95% homology with that which is shown in SEQ ID No.1 and has the biological activity of a nerve growth factor. The mutant in the invention retains the C-terminal integrity in rhNGF by the strategy of expressing rhNGF with 3 amino-acids deleted at C-terminal (rhNGF-D3), so the expression level of rhNGF-D3 protein in eukaryotic cells is increased five- to ten-fold by this modification compared to rhNGF, with the same bioactivity and uniform protein C-terminal.

Description

technical field [0001] The present invention relates to a mutant, specifically a recombinant human nerve growth factor deletion mutant that is highly expressed in a eukaryotic expression system and maintains similar biological activity. Background technique [0002] Nerve Growth Factor (NGF) is the first neurotrophic factor discovered by Levi-Montlcini in mouse sarcoma cells in 1953. NGF can maintain and promote the survival of sympathetic nerves and sensory nerve cells from neural crest, Differentiation and maturation, as well as executive function, are important factors involved in regeneration and functional restoration of injured nerves. [0003] NGF is present in a variety of species and is abundant in tissues of male mouse submandibular gland, bovine seminal plasma, snake venom, guinea pig prostate, and human placenta. The amino acid sequence homology between mouse NGF and human NGF reaches 90%. Mouse NGF extracted from the submandibular gland of mice and human NGF i...

Claims

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Application Information

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IPC IPC(8): C07K14/48C12N15/12C12N15/10C12N15/79C12N5/10
CPCC07K14/48
Inventor 陈薇侯利华宋小红于婷付玲于蕊房婷
Owner 军事科学院军事医学研究院生物工程研究所
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