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A peg-modified immunosuppressive polypeptide of J subgroup avian leukemia virus

An avian leukemia virus and immunosuppression technology, applied in the field of molecular immunology and virology, can solve the problems of mis-panning of endogenous viruses, accelerated virus evolution and mutation, high cost, and reduce direct and indirect economic losses.

Active Publication Date: 2018-08-21
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long purification cycle, high cost, and diversified breeding models, purification has not been effectively implemented in my country at present. Large-scale breeding companies use purification, but the problem of mis-purification of endogenous viruses is serious; unreasonable and unscientific purification measures, it will also accelerate the evolution and mutation of the virus, making the disease more complex

Method used

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  • A peg-modified immunosuppressive polypeptide of J subgroup avian leukemia virus
  • A peg-modified immunosuppressive polypeptide of J subgroup avian leukemia virus
  • A peg-modified immunosuppressive polypeptide of J subgroup avian leukemia virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Artificially synthesized ISU peptide and its identification

[0027] According to the known highly conserved amino acid sequence in the TM protein of representative strains of each subgroup of avian leukosis virus: LQNRAAIDFLLLAQGHGCQDVEGMCCF, the amino acid sequence is shown in SEQ ID NO: 1 in the sequence table, and the ISU peptide shown in the above sequence is artificially synthesized using the prior art . Purified by high performance liquid chromatography (e.g. figure 1 shown) and MS identification (as figure 2 shown), and its purity was identified as more than 97%.

Embodiment 2

[0028] Example 2 Detection of Immune Activity of ISU Peptide

[0029] The lymphocytes needed for the experiment were obtained by aseptically grinding the spleen cells of SPF chickens, and the necessary experimental cells were provided for the ISU immune activity experiment. Lymphocytes derived from the spleen were cultured in a 12-well cell culture plate, and the cell density was adjusted to 1.0×10 after cell counting. 6 / well, divided into 4 groups, group 1 added ISU peptide concentration of 20μg / ml per well, group 2 added 30μl ALV-JNX0101 virus liquid, group 3 added chicken IgG as an irrelevant protein control, the concentration was 20μg per well / ml, in group 4, lymphocytes with normal growth were used as the blank control, and then under the same conditions, each group made 3 parallel controls, at 37°C in 5% CO 2 After 12 hours of cultivation in the incubator, quantitative detection by flow cytometry was performed.

[0030] Flow cytometry detection method

[0031] Coll...

Embodiment 3

[0032] Example 3 Screening of Preparation Conditions for mPEG-ALD Modified ISU Peptides

[0033] 1) pH optimization of the modified buffer system: adjust the pH gradient of the phosphate buffer to 4, 5, 6, 7, 8, 9, and 10, respectively. In a certain proportion, ISU peptide was added. Weigh mPEG-ALD according to 5 times its mass, and weigh an appropriate amount of NaBH 3 CN was added to the reaction system, mixed evenly and detected by SDS-PAGE, the detection results were as follows: Figure 6 As shown, when pH=6, mPEG-ALD had better modification selectivity to ISU peptide, and the modification rate was the highest;

[0034] 2) To optimize the molar ratio of the reactants, mix according to the ratio of ISU:PEG molar mass ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and other reaction conditions According to the initial modification conditions, after 24 hours, samples were taken for SDS-PAGE detection, and the detection results were as follows Figure 7 As shown, the optimal react...

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Abstract

The invention provides a PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide and provides a N-terminal alpha-NH2 PEG modification based immunosuppressive polypeptide (ISU) and a preparation method therefor. Compared with the ISU in the prior art, the PEG modified ISU provided by the invention has the advantages that the duplication of ALV-J can be more remarkably suppressed, a scientific basis and a technical support are provided for the prevention and cure of subgroup J avian leukemia and the preparation of PEG modified polypeptide vaccines, and the economic loss of poultry farming caused by ALV-J infection is effectively reduced.

Description

technical field [0001] The invention relates to the fields of molecular immunology and virology, in particular to a PEG-modified J subgroup avian leukosis virus immunosuppressive polypeptide. Background technique [0002] Avian leukemia of subgroup J is a neoplastic disease caused by avian leukemia virus of subgroup J (ALV-J). Tumors in organs are the main features. The persistent immune response damage leads to frequent secondary infection and mixed infection, which seriously endangers the healthy development of the breeding industry. The follow-up survey on the epidemic status of J subgroup avian leukemia in my country in the past ten years shows that early infection and multiple infections of J subgroup avian leukemia are quite common, showing high infection rate and high incidence rate; multiple / frequent mixed infection; tumor spectrum expansion The pathological changes are increasingly complex, and severe immunosuppression reduces the basic resistance of group diseases...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/15A61P31/14
CPCC07K14/005C12N2740/11011
Inventor 成子强周德方崔熙尧
Owner SHANDONG AGRICULTURAL UNIVERSITY
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