Nucleic acid fragment for encoding rhFGF-6, expression vector, host cell, production method and application

A technology for expressing vectors and nucleic acid fragments, applied in the field of nucleic acid fragments encoding rhFGF-6, which can solve the problems of low activity of rhFGF-6 and difficulty in meeting large-scale applications, so as to promote muscle tissue regeneration, wound healing, and angiogenesis Effect

Active Publication Date: 2017-08-18
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Generally, rhFGF-6 (recombinant human FGF-6, recombinant human FGF-6) is produced by microbial fermentation, but in the prior art, the expression level of rhFGF-6 expressed by microbial fermentation and the activity of the expressed rhFGF-6 are very low , it is difficult to meet the needs of large-scale applications

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  • Nucleic acid fragment for encoding rhFGF-6, expression vector, host cell, production method and application
  • Nucleic acid fragment for encoding rhFGF-6, expression vector, host cell, production method and application
  • Nucleic acid fragment for encoding rhFGF-6, expression vector, host cell, production method and application

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Embodiment 1

[0066] This embodiment provides a nucleic acid fragment encoding rhFGF-6.

[0067] The nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO.1. The sequence is codon-optimized from the natural sequence of human FGF-6 (as shown in SEQ ID NO.2) according to the preference of Escherichia coli, 37 signal peptides are removed, and the secondary structure that is not conducive to expression is eliminated, but it does not change Amino acid sequence of FGF-6.

[0068] The nucleic acid fragment provided in this example is constructed into an expression vector, and the recombinant expression vector is transformed into a host cell to obtain a recombinant bacterium, which is cultured and induced to express with an inducer to obtain high expression of rhFGF-6.

Embodiment 2

[0070] The present embodiment provides a method for producing rhFGF-6, comprising the steps of:

[0071] 2.1 Construction of recombinant expression vector

[0072] Synthesize the rhFGF-6 gene, introduce a start codon and a stop codon at the 5' end and 3' end of the nucleic acid fragment encoding rhFGF-6 (SEQ ID NO.1), and introduce specific enzyme cutting sites NdeI and BamHI.

[0073] The expression vector pET3a and rhFGF-6 genes were treated with NdeI and BamHI double enzyme digestion at 37°C, respectively, and digested for 4 hours.

[0074] The corresponding restriction fragments were recovered and ligated overnight at 16°C with T4 DNA ligase to construct a recombinant expression vector. pET3a-rhFGF-6 recombinant expression vector figure 1 shown.

[0075] 2.2 Transformation and identification of recombinant expression vector

[0076] The recombinant expression vector pET3a-FGF-6 was transformed into the host cell: Escherichia coli BL21(DE3) plysS, positive clones were ...

Embodiment 3

[0118] The present embodiment provides a method for producing rhFGF-6, comprising the steps of:

[0119] Construction of expression vectors, transformation and identification of recombinant expression vectors, cultivation of recombinant bacteria, induction of rhFGF-6 expression, collection and disruption of bacteria, cleaning and denaturation of inclusion bodies are basically the same as the steps and methods provided in Example 2, the difference In this example, the induction culture conditions in this embodiment are: the lactose concentration is maintained at 30 mM, the temperature is 34° C., and the pH is 7.0. In addition, the method for producing rhFGF-6 provided in this embodiment also includes:

[0120] 3.1 Refolding of inclusion bodies

[0121] Take the inclusion body denaturation solution, put it into a dialysis bag with a molecular weight cut-off of 7KD, put in 50 times (v:v) refolding buffer (20mM PB, 2mM EDTA-2Na, 0.1M NaCl , 1% Triton X-100, 5mMDTT, pH7.3), oxidi...

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Abstract

The invention discloses a nucleic acid fragment for encoding rhFGF-6, an expression vector, a host cell, a production method and an application and relates to the field of biological medicines. The nucleic acid fragment for encoding rhFGF-6, provided by the invention, is acquired by optimizing according to a natural sequence of rhFGF-6; 37 signal peptides are removed on the basis of the natural sequence and a secondary structure adverse to expression is eliminated, but an amino acid sequence of rhFGF-6 is not changed; the nucleic acid fragment is constructed onto a suitable carrier so as to form a recombinant expression vector; the recombinant expression vector is converted and then enters the host cell, so that the recombinant bacteria can be acquired; the recombinant bacteria are cultured and an inducer is adopted for inducing expression, so that rhFGF-6 can be highly expressed; the expressed rhFGF-6 has the activities of boosting tissue regeneration, boosting osteogenesis, repairing myocardial damage, boosting wound healing and promoting revascularization.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a nucleic acid fragment encoding rhFGF-6, an expression vector, a host cell, a production method and application. Background technique [0002] Fibroblast Growth Factor-6 (FGF-6) is a member of the FGF family and belongs to the FGF4 subfamily (FGF4, FGF5, FGF6). The human FGF-6 gene is located on human chromosome 12p13 and has three exons of about 15kb in total. The encoded protein consists of a single-chain polypeptide of 208 amino acid residues, and a hydrophobic signal peptide sequence of about 37 amino acid residues at the N-terminus , the mature protein has 171 amino acids (38-208 amino acid residues), its theoretical molecular mass is 19kDa, and its isoelectric point is about 9.73. The amino acid sequence of human FGF-6 has a high homology of 93.3% with that of mouse. Comparative genomics at the mammalian FGF-6 locus revealed that the human, rat and mouse FGF-6 promoters are hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/50C07K1/18C07K1/16A61K38/18A61P21/00A61P19/08A61P9/00A61P17/02C12R1/19
CPCA61K38/00C07K14/50C12N15/70C12N2800/101
Inventor 田海山李校堃郑婕姜潮王申马吉胜许诺
Owner WENZHOU MEDICAL UNIV
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