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Immunoregulation DNA vaccine capable of preventing chicken Eimeria maxima

A DNA vaccine, Eimeria technology, applied in medical preparations containing active ingredients, gene therapy, antibody medical ingredients, etc., can solve the problems of pathogenic threat, high cost, difficult preservation, etc. Strong, long-lasting and thermally stable effects

Inactive Publication Date: 2012-07-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, live vaccines inevitably have disadvantages such as high cost, difficulty in preservation, loose virus and potential disease-causing threats.

Method used

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  • Immunoregulation DNA vaccine capable of preventing chicken Eimeria maxima
  • Immunoregulation DNA vaccine capable of preventing chicken Eimeria maxima
  • Immunoregulation DNA vaccine capable of preventing chicken Eimeria maxima

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Preparation of embodiment 1.Em8 gene

[0038] 1.1 Synthetic primers

[0039] DNAStar software was used to analyze the antigenicity and T cell epitope prediction of the giant Emeria antigen gene EmTFP250 (gene accession number AY239227), and select a sequence with a high antigenic index and a relatively concentrated T cell epitope, which was named Em8. The sequence has 1071 nucleotides (bases 1-1071 of SEQ ID NO.1), encodes 357 amino acids, and is located at positions 6240 to 7310 of the EmTFP250 gene. The primers P1 and P2 of the Em8 gene fragment were designed by using the software primerpremier5.0, and sent to TaKaRa to synthesize the primers. The sequence is as follows:

[0040] P1: 5'-T AAGCTT ATGCGTGAGGACACCGCT-3';

[0041] P2: 5'-A GGATCC CTGAATGTCGCCG-3'

[0042] Among them, the underlined parts are the introduced enzyme cutting sites HindIII and BamHI respectively. To construct recombinant plasmids for tandem cytokines, add coagulation factor X a The...

Embodiment 2

[0053] Example 2. The Construction of Immunomodulatory DNA Vaccine pVAX-Em8-IL-2

[0054] 2.1 Construction of recombinant plasmid pMD18-T-IL-2

[0055] 2.1.1 Primer synthesis

[0056]According to the published nucleotide sequence of chicken IL-2 (chIL-2) (GenBank accession number AF000631), specific primers P3 and P4 were designed using the software primer premier5.0, and sent to TaKaRa to synthesize primers. The sequence is as follows:

[0057] P3: 5'-CT GGATCC ATGTGCAAAGTACTGAT-3';

[0058] P4: 5'-T TCTAGA TTATTTTTGCAGATATCTCAC-3'

[0059] The underlined parts are the introduced enzyme cutting sites BamHI and XbaI respectively.

[0060] 2.1.2 PCR amplification of IL-2 gene

[0061] Add the following components to a thin-walled PCR tube for PCR amplification:

[0062]

[0063] After centrifuging and mixing the above components, denature in a PCR machine at 94°C for 3min; 94°C for 45sec, 60°C for 45sec, 72°C for 45sec, a total of 30 cycles; then extend at 72°C for...

Embodiment 3

[0072] Example 3.RT-PCR detection of transcription of vaccine pVAX-Em8-IL-2 in chickens

[0073] A large number of pVAX-Em8-IL-2 recombinant plasmids were extracted, and 14-day-old chicks (100 μg / bird) were injected intramuscularly into the chest, and the injected site (breast muscle) and non-injected muscle (leg) were collected 7 days later. Total muscle RNA was extracted in one step, DNase I was added to remove residual recombinant plasmid and genomic DNA, and the target gene was amplified by RT-PCR. The results showed that the DNA vaccine was transcribed in chicken muscle cells (see Image 6 ).

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Abstract

The invention relates to an Eimeria maxima DNA vaccine of chicken, belonging to the field of biological veterinary drugs. A gene fragment Em8 of an antigen EmTFP250 at the spore stage of the Eimeria maxima of the chicken is cloned by utilizing the molecular biology technology, and the fragment has higher antigen index and the centralized T cell epitope. The gene fragment is connected with a chicken interleukin-2 (chIL-2) gene in series, thereby constructing a vaccine expression vector pVAX-Em8-IL-2. The vaccine contains a plurality of T cell immune response elements and has the advantages of high safety, long maintenance time in vivo, simple preparation, good thermal stability, convenient storage and transport, time-saving and effort-saving use, and the like. Experiments prove that the immune regulatory DNA vaccine pVAX-Em8-IL-2 has good immune protection effect on the anti-Eimeria maxima infection.

Description

technical field [0001] The invention relates to an immunoregulatory DNA vaccine capable of preventing Eimeria maxima, belonging to the technical field of biological veterinary medicine. The vaccine contains part of the gene of Eimeria maxima sporozoite stage antigen EmTFP250 and chicken interleukin-2 gene (chIL-2), and contains multiple T cell immune response elements. Background technique [0002] Chicken coccidiosis is a kind of parasitic protozoan disease that seriously endangers the production of intensive chicken industry and is prevalent in the world, and the economic loss caused by it is as high as more than 2 billion pounds every year (Williams RB. Acompartmentalized model forestation of the cost coccidiosis to the world's chicken production industry. Int J Parasitol, 1999, 29: 1209-1229). Eimeria maxima (E.maxima) is one of the four common coccidiosis-causing coccidiosis in chickens. Chickens of all ages can be infected, and it mainly occurs in older broilers and l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/012A61K48/00A61P33/02
Inventor 李祥瑞宋小凯严若峰徐立新雷晨昱宋鸿雁
Owner NANJING AGRICULTURAL UNIVERSITY
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