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Deamidated gliadin polypeptide recombinant antigen, recombinant antigen expression gene, recombinant expression vector, preparation method and application thereof

A protein polypeptide, recombinant antigen technology, applied in the direction of fusion polypeptide, chemical instruments and methods, recombinant DNA technology, etc., can solve the problem of serum false negative

Active Publication Date: 2021-07-06
SHENZHEN YHLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serologic testing based on IgA (immunoglobulin A) with the obsolete IgA anti-gliadin antibody (AGA), and later IgA anti-endomysial antibody (anti-EMA) and IgA anti-tissue transglutamate Anti-amidase antibody (anti-tTG), however in IgA-deficient patients, serum anti-tTG and anti-EMA false negatives may occur

Method used

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  • Deamidated gliadin polypeptide recombinant antigen, recombinant antigen expression gene, recombinant expression vector, preparation method and application thereof

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preparation example Construction

[0033] The preparation method of the above-mentioned deamidated gliadin polypeptide recombinant antigen according to an embodiment of the present invention comprises the following steps: obtaining the nucleotide sequence corresponding to the amino acid sequence of the above-mentioned deamidated gliadin polypeptide recombinant antigen, and passing the double-enzyme cutting and ligating into the expression vector to obtain a recombinant expression vector, transfer the recombinant expression vector into a host for expression, and then separate and purify the expression product to obtain a deamidated gliadin polypeptide recombinant antigen.

[0034] In a specific example, the nucleotide sequence corresponding to the amino acid sequence of the above-mentioned deamidated gliadin polypeptide recombinant antigen can be obtained by gene synthesis, and the host can be selected according to the type of expression vector, such as Escherichia coli BL21 (DE3) strain Wait.

[0035] The prese...

Embodiment 1

[0037] 1. Preparation of recombinant antigen of deamidated gliadin polypeptide

[0038] Select DGP dominant epitope peptides, the amino acid sequences are respectively SEQ ID No.1 (named as DGP-1 peptide) and SEQID No.2 (named as DGP-2 peptide), and the amino acid sequence of IL-15 protein is SEQ ID No.3, the amino acid sequence of the hexahistidine tag is SEQ ID No.4, the amino acid sequence of the Flag tag is SEQ ID No.5, and the amino acid sequence of the connecting peptide is SEQ ID No.6. The hexahistidine tag, DGP-1 peptide, interleukin 15, DGP-2 peptide and Flag tag are all connected by connecting peptides and fused into DGP recombinant antigen, the sequence of which is SEQ ID No.7. The DNA of the DGP recombinant antigen was obtained through gene synthesis (General Biosystems (Anhui) Co., Ltd.), and the sequence is SEQ ID No.8. The DNA of the DGP recombinant antigen has an Nde I site upstream and a Hind III site downstream. The DNA was digested with the corresponding r...

Embodiment 2

[0063] The method of this example is basically the same as that of Example 1, the only difference is that pET-28a(+) is selected as the expression vector. Correspondingly, the sensitivity of the prepared detection kit was 0.8 U / mL, the intra-assay and inter-assay differences of the kit were CV=3.65%, CV=4.87%, and the interference met the document standard of NCCLS.

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Abstract

The invention relates to a deamidated gliadin polypeptide recombinant antigen, a recombinant antigen expression gene, a recombinant expression vector and a preparation method and application thereof. The deamidated gliadin polypeptide recombinant antigen includes DGP‑1 peptide, interleukin 15 protein and DGP‑2 peptide, the amino acid sequence of the DGP‑1 peptide is shown in SEQ ID NO.1, and the DGP‑2 The amino acid sequence of the peptide is shown in SEQ ID NO.2. The deamidated gliadin polypeptide recombinant antigen provided by the present invention has good sensitivity, high specificity and low preparation cost when used in the detection of celiac disease, can detect IgG-DGP antibody in serum, and can especially be used for IgA-deficient patients and infants. Detection of patients with potential celiac disease in a young child population.

Description

technical field [0001] The invention relates to the technical field of celiac disease diagnosis, in particular to a deamidated gliadin polypeptide recombinant antigen, a recombinant antigen expression gene, a recombinant expression vector and a preparation method and application thereof. Background technique [0002] Celiac disease (CD), also known as idiopathic steatorrhea, adult celiac disease, nontropical sprue, or glutenous enteropathy, is Chronic autoimmune enteropathy induced by wheat, barley and rye and their products. Genetic studies of celiac disease have shown that the disease is closely related to specific human leukocyte antigen type II genes, such as the HLA-DQ2 and HLA-DQ8 genes located on chromosome 6p21. [0003] Diagnostic methods for celiac disease include genetic testing, intestinal biopsy, and serological testing. Genetic testing is mainly used to screen people at high risk of celiac disease who carry HLA-DQ2 and HLA-DQ8 genotypes. However, due to envir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/68
CPCC07K14/5443C12N15/70G01N33/68C07K2319/21C07K2319/00C07K2319/43G01N2333/5443G01N2800/24C07K14/415G01N33/564G01N2800/06G01N33/54326C12N15/62G01N33/6893
Inventor 郑亮罗春雷钱纯亘胡鹍辉
Owner SHENZHEN YHLO BIOTECH
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