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Immunoloregulation type DNA vaccine capable of preventing Eimeria acervulina

A DNA vaccine and Eimeria technology, applied in the field of DNA vaccines, can solve the problems of poor stability of virulent vaccines and attenuated coccidial vaccines, difficult to control the inoculation amount, etc., and achieve easy storage and transportation, long maintenance time, Prepare simple effects

Inactive Publication Date: 2009-05-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the chicken coccidia vaccines that have been commercialized include live virus vaccines and attenuated vaccines, but the strong virus vaccines and attenuated coccidia vaccines have problems such as poor stability and difficulty in controlling the amount of inoculation.

Method used

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  • Immunoloregulation type DNA vaccine capable of preventing Eimeria acervulina
  • Immunoloregulation type DNA vaccine capable of preventing Eimeria acervulina
  • Immunoloregulation type DNA vaccine capable of preventing Eimeria acervulina

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Preparation of embodiment 1.LDH gene

[0047] 1.1 Synthetic primers

[0048] According to the published nucleotide sequence of LDH (GenBank accession number AY143388), specific primers P1 and P2 were designed using the software primerpremier5.0, and sent to TaKaRa to synthesize primers. The sequence is as follows:

[0049] P1: 5'- GGATCC GCGGTCTTCGAGAA-3';

[0050] P2: 5'- GAATTC GCTGCTGCTTACTTGGAT-3'

[0051] The underlined parts are the introduced restriction sites BamHI and EcoRI, respectively; the boxed part is the initiation codon. 1.2 Extraction of the first generation schizont of E.acervulina

[0052] ① use 10 8 E.acervulina oocysts were infected with coccidia-free chickens, and the duodenum was taken 42 hours later.

[0053] ②Use Hanks solution without calcium and magnesium (NaCl 8.000g, KCl 0.4026g, KH 2 PO 4 0.06g, Na 2 HPO 4 0.0482g, D-glucose 1.0g, NaHCO 3 0.3528g, the pH was adjusted to 7.4, and the fixed solution was 1L) to wash the duod...

Embodiment 2

[0079] The preparation of embodiment 2.DNA vaccine

[0080] 2.1 Construction of recombinant plasmid pVAX-LDH (see Figure 4 )

[0081] The plasmid vectors pMD18-T-LDH and pVAX1 were cloned by double digestion with BamHI and EcoRI, respectively, and the LDH target gene (about 1000bp) and pVAX1 large fragment (about 3000bp) were recovered, and ligated according to an appropriate ratio (molar ratio 1:1). The product was transformed into competent Escherichia coli BL21, the plasmid was extracted, identified by double enzyme digestion with BamHI and EcoRI, and determined to be pVAX-LDH.

[0082] 2.2 Construction of recombinant plasmid pVAX-LDH-IL-2 (see Figure 5-8 )

[0083] 2.2.1 Synthetic primers

[0084] In order to construct the recombinant plasmid of tandem cytokines, use the software primer premier5.0 to design the specific primer P3 without stop codon, add coagulation factor X a The specific hydrolysis sequence encodes the nucleotide sequence (box part), and the underli...

Embodiment 3

[0089] The detection of embodiment 3.DNA vaccine expression situation in chicken body

[0090] The pVAX-LDH and pVAX-LDH-IL-2 recombinant plasmids were extracted respectively, and 14-day-old chicks (100 μg / bird) were injected intramuscularly into the chest, and the injected site (chest muscle) and non-injected site muscle (leg) were collected 7 days later.

[0091] 3.1 RT-PCR detection of DNA vaccine transcription

[0092] Total muscle RNA was extracted in one step, DNase I was added to remove residual recombinant plasmid and genomic DNA, and target genes were amplified by RT-PCR. The results showed that the DNA vaccine was transcribed in chicken muscle cells (see Figure 10 ).

[0093] 3.2 Detection of DNA vaccine translation by Western blot

[0094] 3.2.1 Preparation of recombinant pET28a-LDH protein antiserum

[0095] The pMD18-T-LDH plasmid and the pET28a vector obtained in Example 1 were digested with BamHI and EcoRI, the purified restriction product was recovered, an...

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Abstract

The invention relates to a chicken Eimeria acervulina immunoregulation type DNA vaccine, which belongs to the technical field of biological veterinary medicines. The molecular biology technology is used for connecting lactate dehydrogenase genes of schizont antigen of the first generation of chicken Eimeria acervulina and chIL-2 genes in series to construct a fusion expressing vaccine pVAX-LDH-IL-2. The vaccine comprises a plurality of T cell immune response elements, thus preventing and curing chicken coccidiosis effectively.

Description

technical field [0001] The invention relates to the technical field of biological veterinary medicine, and relates to an immunoregulatory DNA vaccine capable of preventing heap type Eimeria coccidiosis. The vaccine contains Eimeria acervulina lactate dehydrogenase (LDH) gene and chicken interleukin-2 gene (chIL-2). This vaccine contains multiple T cell immune response elements. Background technique [0002] Chicken coccidiosis is a kind of obligate intracellular parasitic protozoan disease, and it is one of the most frequent and most harmful diseases in the chicken industry, and it is difficult to control. Eimeria acervulina (Eimeria acervulina) is One of the important pathogens is only one. So far, the prevention and treatment of chicken coccidiosis mainly relies on the prevention of drugs, but the emergence of problems such as drug resistance and drug residues has greatly restricted the use of anticoccidiosis drugs. Therefore, the use of immune prophylaxis instead of dr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/012A61K48/00A61P33/02
Inventor 李祥瑞徐立新宋小凯严若峰宋鸿雁
Owner NANJING AGRICULTURAL UNIVERSITY
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