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Optimized recombinant antigen polypeptide Abeta1-15-HSP60 nucleotide sequence and high-efficiency preparation method thereof

A technology of nucleotide sequence and recombinant antigen, which is applied in the field of high-efficiency preparation, can solve the problem of low expression of recombinant protein, and achieve the effects of high solubility, low cost and simple purification

Active Publication Date: 2017-02-22
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first technical problem to be solved by the present invention is to provide an optimized recombinant antigen polypeptide Aβ 1-15 - The nucleotide sequence of HSP60 enables efficient and soluble expression in Escherichia coli to overcome the defects of low recombinant protein expression and expression in the form of inclusion bodies in the prior art

Method used

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  • Optimized recombinant antigen polypeptide Abeta1-15-HSP60 nucleotide sequence and high-efficiency preparation method thereof
  • Optimized recombinant antigen polypeptide Abeta1-15-HSP60 nucleotide sequence and high-efficiency preparation method thereof
  • Optimized recombinant antigen polypeptide Abeta1-15-HSP60 nucleotide sequence and high-efficiency preparation method thereof

Examples

Experimental program
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Embodiment 1

[0045] Recombinant Antigen Polypeptide Aβ 1-15 - Optimization of HSP60 nucleotide sequence

[0046] Recombinant antigenic polypeptide Aβ of the present invention 1-15 -The whole gene synthesis of HSP60 is completed by GenScript Biotechnology Co., Ltd. When performing gene synthesis, two enzyme cutting sites, Nde I and Xba I, respectively designed at the upper and lower ends of the target gene, are required to be CGCCATATG and ACGCTCTAGA. The synthesized target gene is 96 bp in full length, loaded into the cloning vector pUC57 plasmid and verified by sequencing. native antigen polypeptide Aβ 1-15 -Compared with the HSP60 gene, the cDNA synthesized by the present invention changes part of the codons into Escherichia coli preferred codons, and at the same time mutates the ACA codons, but does not involve amino acid changes, which is a synonymous mutation.

Embodiment 2

[0048] Recombinant expression vector pCold / Aβ 1-15 - Construction of HSP60

[0049] (1) will carry full-length Aβ 1-15 - The pUC57 and pCold plasmids of HSP60 were respectively digested with Nde I and Xba I, and the two fragments were ligated with T4 ligase to obtain a ligation product.

[0050] (2) Transform the ligation product into Escherichia coli DH5α competent cells by heat stimulation, select clones on the LB plate containing ampicillin, prepare a small amount of plasmids, and screen positive clones by double enzyme digestion and sequencing to obtain pCold / Aβ 1-15 - HSP60 plasmid.

Embodiment 3

[0052] Aβ 1-15 -Construction of HSP60 High Efficiency Soluble Expression Engineering Bacteria

[0053] (1) The recombinant plasmid obtained in Example 2 was transformed into the host bacterium BL21(DE3), which is the engineering bacterium of the present invention.

[0054] (2) Select a single clone on a solid M9 agar medium plate, shake the bacteria overnight in 4mL M9 liquid medium containing 100ug / ml ampicillin at 37°C, 200 rpm.

[0055] (3) Inoculate 1% of the bacterial solution into the M9 culture medium containing 100ug / ml ampicillin, shake the bacteria at 37°C to OD=0.5, add 1mM IPTG at a final concentration of induction, shake the bacteria at 15°C for 6h, and recover the bacteria.

[0056] (4) After the bacteria were disrupted by ultrasound, SDS-PAGE was performed on the whole bacteria, supernatant and precipitated (inclusion body) proteins to analyze the expression of exogenous proteins. Control the changes of exogenous proteins in the bacteria and supernatant before...

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Abstract

The invention discloses an optimized recombinant antigen polypeptide Abeta1-15-HSP60 nucleotide sequence and a method of efficiently solubly expressing in Escherichia coli. The method comprises the following steps: optimizing the recombinant antigen polypeptide Abeta1-15-HSP60 nucleotide sequence, constructing a pCold / Abeta1-15-HSP60expression vector, inducing a foreign protein expression in Escherichia coli with IPTG at low temperature, wherein the quantity of foreign protein expression can achieve more than 50% of total bacterial protein, and the soluble expression quantity achieves more than 90% of total expression quantity. The method can be used for efficiently preparing the recombinant antigen polypeptide Abeta1-15-HSP60.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an optimized recombinant antigen polypeptide Aβ 1-15 -HSP60 nucleotide sequence and its efficient preparation method in Escherichia coli. Background technique [0002] Amyloid β (Aβ) is a transmembrane protein with a molecular weight of about 4KD, which is produced by the hydrolysis of amyloid precursor protein (APP). APP has two metabolic pathways: the first pathway is that α-secretase acts on the 16-17 amino acids of the Aβ sequence of APP, and after hydrolysis, γ-secretase acts on it to produce a smaller fragment p3 and an intracellular fragment (APP intracellular domain, AICD), the product of this pathway produces neurotrophic and neuroprotective effects on nerve cells; the final product of the other pathway is mainly Aβ, and this pathway is that APP produces a large N-terminal after the hydrolysis of its first amino acid sequence by β-secretase Fragment (sAPPβ) and small trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C12N1/21C07K19/00C12R1/19
CPCC07K14/47C07K14/4711
Inventor 王柯朱雪朱凌周凡凡张凯
Owner JIANGSU INST OF NUCLEAR MEDICINE
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