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PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide

An avian leukemia virus, immunosuppressive technology, applied in the fields of molecular immunology and virology, can solve the problems of mis-panning of endogenous viruses, accelerated virus evolution and mutation, complex diseases, etc., and achieve the effect of reducing direct and indirect economic losses

Active Publication Date: 2015-11-25
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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  • PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide
  • PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide
  • PEG (Polyethylene Glycol) modification based subgroup J avian leukemia virus (ALV-J) immunosuppressive polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Synthesis and identification of ISU peptides

[0027] According to the known highly conserved amino acid sequence in the TM protein of each subgroup representative strain of avian leukemia 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 by the prior art. Analyzed and purified by high performance liquid chromatography (such as figure 1 shown) and MS identification (as figure 2 shown), and its purity was identified to be more than 97%.

Embodiment 2I

[0028] Example 2Immune activity detection of ISU peptide

[0029] The lymphocytes required for the experiment were obtained by aseptically grinding the spleen cells of SPF chickens to provide the necessary experimental cells 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 Group 1 was added with ISU peptide at a concentration of 20 μg / ml per well, group 2 was added with 30 μl ALV-JNX0101 virus solution, and group 3 was added with chicken IgG as an irrelevant protein control at a concentration of 20 μg / well per well. ml, group 4 used normal growing lymphocytes as a blank control, and then under the same conditions, each group made 3 parallel controls, at 37°C in 5% CO 2 Quantitative flow cytometry was performed after culturing in the incubator for 12 h.

[0030] Flow cytometry detection method

[0031] Spleen cells were collected, 1 m...

Embodiment 3

[0032] Example 3 Screening of the preparation conditions of mPEG-ALD modified ISU peptide

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

[0034] 2) Optimization of the molar ratio of the reactants, respectively, according to the ratios 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, SDS-PAGE was sampled after 24 hours for detection, and the detection results were as follows Figure 7 As sh...

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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 immunosuppressive polypeptide of J subgroup avian leukemia virus. Background technique [0002] Avian leukemia of subgroup J is a neoplastic disease caused by avian leukemia virus of subgroup J (ALV-J). The main feature is the appearance of tumors in organs. Persistent immune response impairment leads to frequent secondary infections and mixed infections, which seriously endangers the healthy development of aquaculture. A follow-up survey of the epidemic situation of avian leukemia in subgroup J in my country in the past ten years shows that early infection and multiple infection of avian leukemia in subgroup J have become quite common, showing a high infection rate and high morbidity; mixed infection is frequent / frequent; tumor spectrum expansion The pathological changes are increasingly complex, and severe immunosuppression reduces the basic res...

Claims

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Application Information

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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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