Adjuvant-free immunity regulating agent for preventing and treating type 1 diabetes

An immunomodulator, type 1 diabetes technology, applied in DNA recombination technology, protein separation and purification technology and medical related fields, can solve the problems of loss of drug activity, limited application prospects, high cost, etc.

Inactive Publication Date: 2009-11-11
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for peptide drugs, due to the presence of a large number of peptidolytic enzymes and proteolytic enzymes in the gastrointestinal tract, oral administration is easily catalyzed and decomposed, and the drug activity is lost; or the first-pass effect occurs and eliminated by liver metabolism
For the generation and maintenance of oral tolerance, a large amount of antigen must be orally administered as early as possible and for a long time. If the source of antigen is limited or the cost is too high, it will limit its broad application prospects

Method used

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  • Adjuvant-free immunity regulating agent for preventing and treating type 1 diabetes
  • Adjuvant-free immunity regulating agent for preventing and treating type 1 diabetes
  • Adjuvant-free immunity regulating agent for preventing and treating type 1 diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Design, synthesis and cloning of CTB-GADIII polypeptide gene

[0047] According to the amino acid sequence of CTB gene and GAD65 antigen peptide gene, Escherichia coli preferred codons were selected, and six oligonucleotide fragments were designed by computer, and GADIII polypeptide gene was first synthesized by PCR method, and the gene was cloned into the downstream of pET28a-CTB gene , transformed into Escherichia coli to obtain a recombinant plasmid named pET28a-CTB-GADIII. The specific method is as follows:

[0048] Six oligonucleotides:

[0049] P1: 5′-AAA GCTAG CGGTGGTGAATACGTTACTCTGAAAAAAATGCGTGAAATCATCGGTT-3′

[0050] P2: 5′-AAAAAGCTTAGTCACCTGAACCACCCGGCCAACCGATGATTTCACGCATT-3′

[0051] P3: 5′-GATAACCGGAGCAACTTTAGACAGACGTGAGTCACCTGAACCACCCGGCCAA-3′

[0052] P4: 5′-AAAAAGCTTACATCATACGAGCTTTGATAACCGGAGCAACTTTAGAC-3′

[0053] P5: 5′-AACGGAGTCAGTACCGATACCCAGAGCAGCATCATACGAGCTTTGATAA-3′

[0054] P6: 5′-AAA AAGCTT ATTCGTCGCATTTGATTAAGATAACGGAGT...

Embodiment 2

[0058] Embodiment 2: Expression of CTB-GADIII gene in Escherichia coli

[0059] The recombinant plasmid pET28a-CTB-GADIII was transformed into Escherichia coli BL21. Pick a single colony from a plate with different transformants and inoculate it into LB liquid medium containing 50 μg / ml kanamycin. After culturing at 37°C for 4 hours, add α-lactose at a final concentration of 5 mmol / L to induce E. coli to express T 7 RNA polymerase, continue culturing to express the fusion protein CTB-GADIII. After induction, a small amount of bacterial liquid was taken every 1 hour, and the bacterial cells were recovered by centrifugation. SDS-PAGE electrophoresis and thin-layer scanning showed that the fusion expression of the CTB-GADIII polypeptide gene had been realized. The fusion protein reaches a stable maximum expression level after 6 hours of induction, and the fusion protein accounts for more than 50% of the total bacterial protein. The results are shown in image 3 .

Embodiment 3

[0060] Embodiment 3: Separation and purification of recombinant protein CTB-GADIII

[0061] Inclusion bodies were washed 3 times with PBS buffer containing 1% Triton. The initially purified fusion protein CTB-GADIII was dissolved in 0.1 mol / L Tris buffer solution (pH 8.9) containing 8 mol / L urea. The denatured protein was isolated and purified using CM52 cation exchange resin. It was eluted with a Tris / NaCl gradient containing 8 mol / L urea, collected in sections for SDS-PAGE electrophoresis detection, and CTB-GADIII was in the elution peak at 120-150 mmol NaCl. The obtained purified protein was dissolved in the renaturation buffer containing 0.1mol / L Tris-HCl, pH 8.9, 0.4mol / L NaCl and 2mmol / l EDTA, and incubated overnight at 37°C for renaturation. The refolded protein was further separated from monomers and pentamers by molecular sieves. Check its purity by SDS-PAGE electrophoresis, see Figure 4 .

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Abstract

The invention provides an adjuvant-free immunity regulating agent for preventing and treating type 1 diabetes. Polypeptide with single antigen is hard to re-establish immunological tolerance and block self-immune attack after an organism has received the self-immune attack. Three antigen epitope polypeptide genes (stemmed from a human glutamate decarboxylase 65 molecule) are orderly connected in series and connected to a lower reach of a cholera toxin B subunit gene so as to realize the fusional expression of the serially connected antigen epitope polypeptides and CTB. Without adding any adjuvant, the obtained fusion protein can excite the organism to generate the immunological tolerance aiming at body fluid of the serially connected antigen polypeptides by a way of mucous membrane immunity and obviously lower the occurrence of the NOD mouse diabetes, so that the immunity regulating agent can be used for preventing and treating the type 1 diabetes.

Description

technical field [0001] The invention relates to DNA recombination technology, protein separation and purification technology and medical related fields. More specifically, the present invention relates to the construction, expression and separation and purification of fusion protein CTB-GADIII. In the medical field, the present invention is an immunomodulator, which can be used for preventing and treating type 1 diabetes. Background technique [0002] Diabetes is one of the most common chronic diseases. With the improvement of people's living standards, the aging population and the increase in the incidence of obesity, the incidence of diabetes is increasing year by year. Diabetes is divided into type 1 and type 2. Type 1 diabetes is an autoimmune disease, manifested by the body's immune intolerance to pancreatic antigen components, and autoimmune destruction of pancreatic β cells, resulting in loss of pancreatic function, reduced or absent insulin secretion, and metaboli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/385A61K39/39A61P3/10
Inventor 刘景晶王华倩孙云霄熊琪琰杨洁金亮李建平李泰明曹荣月吴洁
Owner CHINA PHARM UNIV
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