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Protein vaccine against tumor necrosis factor alpha and use thereof

A vaccine and fusion protein technology, applied in the field of biomedicine, can solve the problems of high cost, achieve low therapeutic dose, improve immune effect, and inhibit inflammatory response

Active Publication Date: 2020-04-28
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, hemocyanin is a highly glycosylated protein with extremely large molecular weight. Although it has high immunogenicity, it cannot be recombined or synthesized in vitro, and can only be purified by keyhole limpet. Therefore, higher cost

Method used

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  • Protein vaccine against tumor necrosis factor alpha and use thereof
  • Protein vaccine against tumor necrosis factor alpha and use thereof
  • Protein vaccine against tumor necrosis factor alpha and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Obtaining of TNF-α mutein of the present invention

[0035] Using computer-aided design, combined with previous studies of the present invention, it is found that the active center of hTNF-α includes four domains of 29-36AA, 84-91AA, 117-119AA and 143-148AA. Among them, amino acid residues such as Glu23, Leu29, Arg32, Ala33, Asn34, Leu75, Ala84, Tyr87, Asp143 may be important amino acids involved in the biological activity of hTNF-ɑ.

[0036] The present invention designs mutant proteins for the following sites, and according to the sequence homology comparison of human and mouse TNF-α proteins, designs corresponding mouse mutant proteins for preclinical research (specific mutation sites and mutation methods As shown in Table 1). On this basis, the present invention synthesizes the gene sequence encoding the above-mentioned mutant protein through conventional nucleic acid synthesis technology, and uses this as a template to carry out PCR amplification thro...

Embodiment 2

[0046] Example 2: Identification of Biological Activity of TNF-α Mutants

[0047] The identification of the biological activity of TNF-α mutants is determined by the activity test of L929 cells. L929 cells are mouse fibroblasts sensitive to TNF-α activity. This method is a standard method for measuring the biological activity of TNF-α protein. The brief description is as follows : L929 in logarithmic growth phase at 1.5×10 4 per well in a 96-well plate. Design 10 dose gradients, each dose gradient has 2 duplicate wells, the actual dilution concentration of TNF-α mutant or standard control substance is 1.0×10 -6 mg / ml. Each protein was diluted in complete medium PRMI 1640 containing 3% FBS and 0.7 μg / ml actinomycin D. After culturing at 37° C. for 24 hours, the number of surviving cells was detected by MTT method, and a curve was drawn to determine the biological activity of each mutant protein at the level of 50% effective concentration (IC50).

[0048] The result is as f...

Embodiment 3

[0049] Example 3 Y86K breaks immune tolerance and produces neutralizing antibodies

[0050] Female Bab / c mice aged 6-8 weeks were randomly divided into 6 groups: wild-type protein immunization group, mutant protein immunization group (mTNF-mL, mTNF-mR, mTNF-mA, mTNF-mL2, mTNF-mY, mTNF-mD), 6 rats in each group. In this study, Al(OH) 3 as an adjuvant. Add 10μg protein to 50μl 50mM PB, add Al(OH) in equal volume 3 50μl (concentration: 10mg / ml), mix thoroughly. Mice were immunized subcutaneously on both sides of the groin, once every other week, a total of 6 times. Before the first immunization and 1 week after each immunization, blood was collected from the orbital canthus venous plexus of the mice, and the serum was separated. Anti-mouse TNF-α antibody titers in serum were detected by ELISA. The result is as figure 2 Shown, compared with wild-type protein, each mutant vaccine all can stimulate mice to produce higher antibody titer, wherein mTNF-mY (amino acid sequence ...

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Abstract

The invention belongs to the field of biological medicines, and relates to a mutant protein vaccine aiming at tumor necrosis factor alpha (TNF-alpha) and applications of the mutant protein vaccine. The invention aims at providing the protein vaccine which is good in property and takes TNF-alpha as the target spot. According to the scheme, by immunizing the TNF-alpha mutant protein vaccine, the biological activity of the TNF-alpha mutant protein vaccine is inhibited, the cross immunologic reaction is generated in vivo, the biological functions of TNF-alpha are neutralized, and the autoimmune diseases and inflammatory diseases are inhibited. The protein vaccine aiming at TNF-alpha can be used for treating the autoimmune diseases and the inflammatory diseases and preventing the relapse of the autoimmune diseases and the inflammatory diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a protein vaccine against tumor necrosis factor alpha (TNF-alpha) and its application. Background technique [0002] Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. Traditional treatment drugs for autoimmune diseases mainly include non-specific anti-inflammatory drugs (such as 5-aminosalicylic acid, glucocorticoids) and immunosuppressants (such as azathioprine, cyclosporine). However, due to the serious side effects of these drugs, many patients cannot tolerate long-term use and affect the curative effect. [0003] Tumor necrosis factor α (TNF-α) is one of the important molecules in the regulation of immune and inflammatory responses, involved in a variety of disease types, including rheumatoid arthritis, inflammatory bowel disease, endotoxic shock, cerebral malaria and graft resistance host response. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/385A61K39/39A61K39/00C07K19/00A61P29/00A61P19/02A61P17/06A61P1/00A61P19/08
CPCA61K39/0008A61K39/385A61K39/39A61K2039/55505A61K2039/6037C07K14/525C07K2319/40
Inventor 万洋何谷杨莉罗彦魏于全
Owner SICHUAN UNIV
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