Method for preparing antigen substitute by using human fibronectin type III domain to display two antigenic epitopes

A fibronectin and domain technology, which is applied in the field of preparation of antigen substitutes based on the display of double antigenic epitopes of human fibronectin type III domain, can solve the problem of difficulty in antigen preparation, and achieve efficient preparation, short cycle and easy operation. Effect

Inactive Publication Date: 2018-06-19
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of difficult preparation of protein antigens, the present invention provides a method for preparing antigen substitutes ...

Method used

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  • Method for preparing antigen substitute by using human fibronectin type III domain to display two antigenic epitopes
  • Method for preparing antigen substitute by using human fibronectin type III domain to display two antigenic epitopes
  • Method for preparing antigen substitute by using human fibronectin type III domain to display two antigenic epitopes

Examples

Experimental program
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Embodiment 1

[0030] The construction of recombinant protein expression vector displaying NT-proBNP double epitope of human fibronectin type Ⅲ domain:

[0031] Replace the base sequence encoding the Fn3FG loop region with the base sequence encoding the NT-proBNP12-21 amino acid residue epitope (ie, the base sequence encoding Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys) , replacing the base sequence encoding the 3' end of Fn3 with the base sequence encoding the epitope of amino acid residues 62-73 of NT-proBNP, so as to realize the stable expression of the NT-proBNP dual epitope gene through the human fibronectin type III domain method. Introduce the base sequence encoding SSNNNNNNNNNNN (S represents serine residue, N represents asparagine residue) short peptide base sequence in encoding two short peptide base sequences, to prolong the distance between two antigenic epitopes, reduce two The interaction between them; the base sequence encoding 6 groups of amino acid residues is introduced at the 5' ...

Embodiment 2

[0034] Escherichia coli BL21(DE3) transformation:

[0035] 0.1 μg of the recombined plasmid pET28-rFn3-2-epitopes was heat-shocked to transform 100 μL of E. coli BL21 (DE3) chemically competent cells. The specific operation is: after gently mixing the above plasmids and chemically competent cells, ice bath for 3 minutes, then heat shock at 42°C for 80 seconds, ice bath for 5 minutes, and then add 200 μL of SOC medium preheated at 37°C; 37 After standing in the incubator for 30 minutes, culture at 37°C and 220rpm for 1 hour with shaking; take 50 μL and 100 μL of cultured bacterial liquid to gradient plate respectively, and the culture medium used is the antibiotic containing kanamycin (final concentration: 50 μg / mL). LB solid medium. Incubate overnight in a 37°C incubator to grow positive single clones.

[0036] Among them, the formula of LB solid medium is: tryptone (Tryptone) 10g / L, yeast extract (Yeasextract) 5g / L, sodium chloride (NaCl) 10g / L, agarose powder 15g / L, and dd...

Embodiment 3

[0038] Induced expression of human fibronectin type III domain displaying NT-proBNP dual epitope recombinant protein:

[0039] The positive transformants obtained in Example 2 were picked and streaked on LB solid medium containing kanamycin antibiotic (final concentration: 50 μg / mL), and cultured overnight in an incubator at 37°C. Single clones were picked and inoculated in 3 mL LB liquid medium containing kanamycin antibiotic (final concentration: 50 μg / mL), and cultured overnight at 37° C. and 220 rpm. The next day, inoculate the bacterial solution into 500 mL LB liquid medium containing kanamycin antibiotic (final concentration 50 μg / mL) at a ratio of 1:100, and cultivate to OD at 37 ° C and 220 rpm 600nm reach 0.5. Then, isopropylthiogalactopyranoside (IPTG) was added with a final concentration of 0.2 mM, and the expression was induced at 26° C. and 220 rpm for 4 hours. During this process, the recombinant protein rFn3-2-epitopes was expressed intracellularly in Escheric...

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Abstract

The present invention discloses a method for preparing an antigen substitute by using human fibronectin type III domain (Fn3) to display two antigenic epitopes. According to the method, through gene recombination, base sequences for encoding two antigenic epitopes are respectively recombined at the FG loop region and the 3' terminal of the sequence encoding Fn3 gene to from an Fn3 and double antigenic epitope fusion gene; and the fusion protein displaying the double antigenic epitope is efficiently and rapidly expressed and prepared with an Escherichia coli expression system. According to thepresent invention, the antigen substitute displaying the double antigenic epitopes can be simply, rapidly and efficiently prepared so as to establish the technical platform for the production of the corresponding immunological assay.

Description

technical field [0001] The invention belongs to the field of genetic engineering and protein engineering, and in particular relates to a method for preparing an antigen substitute based on the display of double antigenic epitopes by the human fibronectin type III domain. Background technique [0002] Human fibronectin type III domain (Fn3 for short) is a kind of skeleton protein that can tolerate multiple amino acid insertions, deletions or substitutions while maintaining its folding and tertiary structure. It has the characteristics of high stability and high solubility. Can be expressed at high levels in E. coli. As a small independent folding unit, Fn3 does not contain cysteine ​​and disulfide bonds, and consists of four β-sheets and an α-helix; three loop regions (BC loop region, DE loop region and FG loop area). A large number of studies have shown that the FGloop region has high flexibility, and its mutation or deletion has little effect on the structure and stabilit...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C12N1/21G01N33/58G01N33/68C12R1/19
CPCC07K14/78C12N15/70G01N33/581G01N33/68
Inventor 胡学军阮瑶丁宁付鑫杨春光张伟峻
Owner DALIAN UNIV
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