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Type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 and application thereof

A type 1 diabetes, scfv-c22 technology, applied in the field of biomedicine, can solve the problems of difficult to penetrate the membrane, no anti-type 1 diabetes ZnT8-specific single-chain antibody, and large molecular weight.

Active Publication Date: 2016-11-09
张梅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Antibody research has a history of more than 100 years. Kohler and Milstein founded B lymphocyte hybridoma technology in 1975. Mouse-derived monoclonal antibodies are widely used in the diagnosis and treatment of diseases, but because mouse-derived monoclonal antibodies are immunogenic to humans , easy to produce human anti-mouse antibody (human anti-mouse antibody, HAMA and hypersensitivity reactions, and because of its large molecular weight, it is difficult to penetrate membranes and other shortcomings, which limits its application in the field of disease treatment
However, there is no relevant report on anti-type 1 diabetes ZnT8-specific single chain antibody

Method used

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  • Type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 and application thereof
  • Type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 and application thereof
  • Type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction and expression of embodiment 1 recombinant ZnT8 prokaryotic expression plasmid

[0036] Design primers ZF and LR (Table 1), use the ZnT8 plasmid (purchased from Beijing Yiqiao Sino Biotechnology Co., Ltd., HG11621-M) as a template to amplify the human ZnT8 amino terminal (1-74aa) gene, and design primers LF and CR (Table 1), use the ZnT8 plasmid as a template to amplify the human ZnT8 carboxy-terminal (269-368aa) gene, and use primers ZF and CR to construct an aminocarboxyl fusion gene by overlapping after gel recovery of the PCR product.

[0037]Referring to SEQ ID No.55 in the US9023984 (B2) sequence list, design the ZnT8 carboxy-terminal dimerization mutant gene, as shown in SEQ ID No.7, and entrust the biological company to synthesize it. The dimer contains 2 human ZnT8 carboxy-terminal genes , the first amino acid at 325 is arginine (Arg), and the second is tyrosine (Trp) at 325. In this dimer, two human ZnT8 carboxy-terminal genes are linked by a flex...

Embodiment 2T1D

[0046] Example 2 Construction of T1D scFv phage antibody library

[0047] The experimental samples were the peripheral blood of 50 volunteers with incipient T1D. Among them, 31 volunteers were positive for serum ZnT8 antibody. Serum ZnT8 antibody levels were detected by radioimmunoligand method (see Gu Y, Zhang M, Chen H, Wang Z, Xing C, Yang H, et al. Discordant association of islet autoantibodies with high-risk hla genes in chinese type for detailed methods 1diabetes.Diabetes / metabolismresearch and reviews 2011;27:899-905). All patients signed the informed consent. The peripheral blood PBMCs of T1D volunteers were sorted. Extract the PBMC RNA from the peripheral blood of T1D volunteers, and then synthesize cDNA by reverse transcription. The specific operation steps are carried out according to the instructions. By RT-PCR method, use human single-chain antibody (scFv) universal primers (Table 2) to amplify the heavy chain and light chain, link them into scFv by overlap PC...

Embodiment 3Z

[0062] Example 3 Characteristic Analysis of ZnT8-specific scFv

[0063] Western blot detection: transfer the purified recombinant ZnT8 aminocarboxyl fusion protein to PVDF membrane after 12% protein gel electrophoresis, block with 5% milk, use purified scFv as the primary antibody, and HRP-labeled HA antibody as the secondary antibody to detect Binding activity of scFv-C22 to ZnT8. The results showed that scFv-C22 could detect the aminocarboxyl fusion protein, and obvious bands could be seen at about 20kD, which was in line with expectations, while no bands were seen in the negative control, indicating that scFv-C22 had good binding ability to ZnT8 protein ( Figure 6 ).

[0064] Affinity detection: completed by Suzhou Baituo Biotechnology Co., Ltd. Affinity results showed that scFv-C22 had a high affinity with ZnT8 aminocarboxyl fusion protein, with an affinity of 5.953×10 -8 kD(M)( Figure 7 ).

[0065] ELISA detection: The purified ZnT8 carboxy-terminal dimerization mu...

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Abstract

The invention discloses a type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 and application thereof. Amino acid sequences of the type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 are shown as SEQ ID NO.5, and coding gene sequences of the type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 are shown as SEQ ID NO.6. The type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 and the application have the advantages that T1D scFv phage libraries are successfully established, and the type-1 diabetes mellitus resistant ZnT8 specific single-chain antibody scFv-C22 is screened and authenticated and can be used for diagnosing and treating type-1 diabetes mellitus and / or preparing prognosis reagents.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C22 and application thereof. Background technique [0002] Zinc transporter (ZnT) is a group of transmembrane proteins that transport zinc ions from intracellular to extracellular, and maintain the balance of intracellular and extracellular zinc ions together with ZIP family proteins. In 2004, Cheimienti et al first discovered the ZnT8 protein in the ZnT family, which is encoded by the SLC30A8 gene and is highly specifically expressed in the pancreas. Further research found that ZnT8 is specifically expressed on the islet β cells and overexpressed in the INS-1 cells ZnT8 can promote the aggregation of zinc ions and enhance insulin release after glucose stimulation. These studies indicated that ZnT8 plays an important role in the synthesis and secretion of insulin. [0003] Type 1 diabetes (T1D) is an organ-speci...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/53A61K39/395A61P3/10
CPCA61K2039/505C07K16/18C07K2317/56C07K2317/622
Inventor 张梅张晓杨涛吴倩陈恒秦瑶
Owner 张梅
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