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Method for producing secretion expression recombinant human fibroblast growth factor-21

An expression vector and host cell technology, applied in the field of biomedicine, can solve the problems of low product recovery rate, protein damage, affecting protein activity, etc., and achieve the effects of simple purification process, simplified purification process, and improved yield

Active Publication Date: 2009-03-04
李校堃 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The product recovery rate obtained by this process is very low, which also affects the protein activity
At present, foreign studies have shown that the expression of FGF21 mainly exists in the form of inclusion bodies, which brings difficulties to medical research and drug development
[0008] At present, the production process of the FGF21 Escherichia coli expression system in the world, whether the expression product exists in the form of soluble protein or not, is expressed in the cytoplasm, and the protein must be broken by ultrasonic waves to release the protein, which not only damages the protein itself role, but also increased the difficulty and cost of purification

Method used

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  • Method for producing secretion expression recombinant human fibroblast growth factor-21
  • Method for producing secretion expression recombinant human fibroblast growth factor-21
  • Method for producing secretion expression recombinant human fibroblast growth factor-21

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Construction of engineering bacteria expressing rhFGF-21

[0060] 1. Synthesis of the target gene

[0061] According to the amino acid sequence of the FGF21 gene with the signal peptide removed, primers were designed, and the FGF21 gene with the signal peptide removed was obtained by complementary extension. Using the hEGF gene as a blueprint, according to the degeneracy of codons, the restriction sites used in this experiment were eliminated, and the online software Primer Finder and DNA STAR were used to design FGF21 primers, a total of 14 (see the sequence list for specific primer sequences, SEQ ID NO.2-15), each pair contains a 20bp complementary sequence, wherein a NcoI restriction site is introduced at the 5' end and an XhoI restriction site is introduced at the 3' end. The FGF21 gene was synthesized by bridge PCR. The specific synthesis method is as follows:

[0062] PCR system one:

[0063]

[0064]

[0065] PCR system two:

[0066]

[00...

Embodiment 2

[0083] Example 2 Selection of suitable expression vectors

[0084] According to the method similar to Example 1, the FGF-21 sequence was inserted into several different expression vectors, such as pET20b(+), pET22b(+), pET26b(+) and pET27b(+) (all purchased from Invitorgen) Using the same enzyme cutting sites (NcoI / XhoI), plasmids pET20b(+)-rhFGF-21, pET26b(+)-rhFGF-21, and pET27b(+)-rhFGF-21 were obtained.

[0085] Plasmids pET20b(+)-rhFGF-21, pET26b(+)-rhFGF-21, pET27b(+)-rhFGF-21.

[0086] The corresponding host bacteria were transformed respectively, and the resistant strains were selected. Shake flask test with engineering bacteria containing plasmid pET22b(+) / rhFGF-21, after 2 hours of induction with IPTG (or arabinose), the expression of the target protein expressed by engineering bacteria containing plasmid pET22b(+)-rhFGF-21 The amount accounts for about 10% of the total protein, and the expression amount of the target protein expressed by the engineered bacteria co...

Embodiment 3

[0088] The selection of embodiment 3 best culture medium

[0089] Pick the Escherichia coli W3110 (hereinafter referred to as "W3") monoclonal that is transferred into pET22b(+)-rhFGF-21 prepared in Example 1, inoculate it into a 250ml Erlenmeyer flask containing 50ml LB seed solution, and cultivate it for 3-4hr , to be OD 600 reach 0.8-1.0, inoculate in a 1000ml Erlenmeyer flask containing 250ml of improved seed solution at a ratio of 1:10, cultivate for about 8-10h, and wait until OD 600 When it reaches 6-8, put it into the tank for fermentation according to the ratio of 1:10, add carbon source (glucose or glycerin), magnesium salt, and adjust pH to 6.8-7.0 with ammonia water, temperature at 37°C, DO>30%, wait for OD 600 When it reaches 15-18, add 1mM IPTG to start the induction, add carbon source (glucose or glycerol), nitrogen source, and phosphate buffer to adjust the pH to 7.2-7.4, DO>50%, temperature 32°C, and induction for 5 hours to end. The samples were detected by...

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Abstract

The invention provides a method for producing FGF21. The invention first designs and synthetizes full-length FGF21 nucleotide sequence which is integrated with secreted signal peptide to be structured into an expression carrier capable of secreting and expressing FGF21 protein; the expression carrier is led into an appropriate host cell; under the guidance of the secreted signal peptide, FGF21 is expressed in cell periplasm, so that the purification procedure is convenient, the finished product ratio is improved and the mass production of rhFGF-21 becomes possible.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to an optimized method for producing human fibroblast growth factor-21 (FGF21), expression vectors and host cells used in the method. Background technique [0002] Fibroblast growth factor (FGF) is a class of structurally related proteins encoded by members of the FGF gene family. At present, 23 members have been found, and their central regions all contain an amino acid sequence with a homology of 30%-70%. The members of the large FGF family, as intercellular multifunctional signaling molecules, regulate various physiological functions of organisms. Many members of the FGFs family are closely related to the occurrence, development and metastasis of tumors. [0003] FGF21, a new member of the FGF family, is a secretable protein. First isolated from mouse embryos, the human FGF21 cDNA encodes 209 amino acids, which has about 75% homology with the mouse FGF21 amino acid. The amino acid se...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C12N1/21C12P21/04C12R1/19
Inventor 李校堃王会岩肖业臣田海山赵宏鑫张耀方万晓姗解佳森杨苹
Owner 李校堃
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