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Solubleand efficient recombinant expression methodfor fibroblast growth factor protein

A technology for fibroblasts and growth factors, applied in the field of recombinant proteins, can solve the problems of difficulty in obtaining efficient and stable expression of fibroblast growth factor proteins, and achieve the effects of being conducive to popularization and application, good prospects and economic value, and a simple method.

Pending Publication Date: 2019-04-16
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a method for soluble and high-efficiency recombinant expression of fibroblast growth factor protein, which solves the technical problem that it is difficult to obtain high-efficiency and stable expression of fibroblast growth factor protein in the Escherichia coli system

Method used

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  • Solubleand efficient recombinant expression methodfor fibroblast growth factor protein
  • Solubleand efficient recombinant expression methodfor fibroblast growth factor protein
  • Solubleand efficient recombinant expression methodfor fibroblast growth factor protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of recombinant FGF7 expression strain.

[0043] According to the human FGF7 amino acid sequence provided by the NCBI database (sequence SEQ ID NO: 1), and combined with the E. coli code preference principle, the cDNA sequence encoding the amino acid of the FGF7 variant was designed, and the FGF7 variant containing the coded FGF7 variant was obtained through full gene synthesis DNA fragments. The DNA fragment encoding the FGF7 variant obtained from the whole gene, and the pET-3c vector plasmid, which is the expression vector regulated by the T7 promoter, were digested with Nde I+BamH I and ligated by DNA polymerase to obtain Circular recombinant plasmid encoding FGF7 variant. Transform the obtained recombinant plasmid into BL21(DE3)plysS competent cells, spread it on an LB agarose plate containing 100ug / ml ampicillin and 25ug / ml chloramphenicol resistance, and screen out monoclonal colonies to obtain expression recombinant The engineered strain of ...

Embodiment 2

[0044] Example 2: Construction of recombinant FGF21 expression strain.

[0045] According to the human FGF21 amino acid sequence (SEQ ID NO: 2) provided by the NCBI database, combined with the E. coli code preference principle, a cDNA sequence encoding FGF21 amino acid was designed, and a DNA fragment encoding FGF21 was obtained through full gene synthesis. The DNA fragment encoding FGF21 obtained from the whole gene, and the pET-30a vector plasmid, which is a T7 promoter-regulated expression vector, were digested with Nde I+BamH I and ligated by DNA polymerase to obtain FGF21 The circular recombinant plasmid. The obtained recombinant plasmid pET-30a-FGF21 plasmid was transformed into BL21(DE3)plysS competent, and spread on LB agarose plate containing 50ug / ml kanamycin and 25ug / ml chloramphenicol resistance, and screened out Monoclonal colonies were used to obtain engineered bacteria expressing recombinant FGF21, named pET-30a-FGF21 / BL21(DE3)plysS.

Embodiment 3

[0046] Example 3: Construction of recombinant FGF18 expression strain.

[0047] According to the human FGF18 amino acid sequence provided by the NCBI database (SEQ ID NO: 3), combined with the E. coli code preference principle, a cDNA sequence encoding FGF18 variant amino acids was designed, and a DNA fragment encoding FGF18 was obtained through full gene synthesis . The DNA fragment encoding FGF18 obtained from the whole gene, and the pET-30a vector plasmid, which is a T7 promoter-regulated expression vector, were digested with Nde I+BamH I and ligated by DNA polymerase to obtain a DNA fragment containing encoding FGF18 Variant circular recombinant plasmid. The obtained recombinant plasmid pET-30a-FGF18 plasmid was transformed into BL21(DE3) competent, and spread on the LB agarose plate containing 50ug / ml kanamycin and 25ug / ml chloramphenicol resistance, and the single The colony was cloned to obtain an engineered strain expressing recombinant FGF18, named pET-30a-FGF18 / BL21(D...

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Abstract

The invention discloses a solubleand efficient recombinant expression methodfor fibroblast growth factor protein. In the method, as for an escherichia coli strain of a carrier containing coding-recombinant fibroblast growth factor family protein, expression of intracellularactive protein is promoted by adding a certain dosage of monosaccharide during induction.The fibroblast growth factor family protein comprises FGF1, FGF2, FGF4, FGF7, FGF8, FGF9, FGF11, FGF15, FGF18, FGF21, FGF23 and mutants of the fibroblast growth factor family protein. As for the process that the certain dosage of monosaccharide is added during induction, the monosaccharide is supplemented into before an inductive agent is added, or the monosaccharide is supplemented into while an inductive agent is added, or the monosaccharide is supplemented into after the inductive agent is added. The technology prejudice that expression of lac and araoperons is inhibited by glucose in escherichia colirecombinant is overcome inthe technical field, and the method capable of efficiently promoting expression of the fibroblast growth factor protein in escherichia coli is applied.

Description

Technical field [0001] The invention relates to the field of recombinant proteins, in particular to a method for soluble and high-efficiency recombinant expression of fibroblast growth factor protein. Background technique [0002] Fibroblast Growth Factor (FGF) is a class of structurally related proteins encoded by members of the Fgf gene family. It has effects on endothelial cells, fibroblasts, smooth muscle cells and other mesodermal and neuroectoderm-derived cells. Promote DNA synthesis and cell division, mainly secreted by fibroblasts, endothelial cells, osteocytes, and inert cells. The relative molecular weight is generally 17~34kD (Itoh N et al., 2004, Trends in Genetics, 20(11):563-569 ). [0003] Fibroblast growth factor contains 23 members and can be arranged into 7 subfamilies, each of which contains 2-4 members. The Fgf1, Fgf4, Fgf7, Fgf8 and Fgf9 subfamily genes encode secreted classic FGFs, which use heparin as a cofactor to bind and activate the fibroblast growth fa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/19
CPCC07K14/50
Inventor 谭长城杨辉李金星何勇曹宇范开
Owner CHONGQING PEG BIO BIOTECH CO LTD
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