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Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis

A DNA vaccine, Eimeria technology, applied in the field of DNA vaccines, can solve the problems of high cost, not fully applicable to laying hens and broilers, and poor stability

Inactive Publication Date: 2006-07-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The use of highly toxic vaccines and attenuated coccidia vaccines is a means of prevention, but the highly toxic vaccines are only suitable for breeders, not completely suitable for layer and broiler chickens. Although broiler chickens can use attenuated coccidia vaccines, they have poor stability. The inoculation amount is difficult to control and the cost is high, so that these coccidia vaccines cannot be well developed and utilized

Method used

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  • Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis
  • Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis
  • Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of embodiment 1.pEtK2 gene:

[0043] 1. Synthesize primers P1 and P2, the sequences are:

[0044] The following primers were designed according to the published nucleotide sequence of pEtK2 (Genebank accession number AY389513):

[0045] P1: 5'-AA GGATCC CCAGCAGCGTCCAA-3′

[0046] P2: 5'-GAA GAATTC CGCTGCGGTATCTCCG-3'

[0047] The underlined parts are the introduced restriction sites BamHI and EcoRI, respectively; the boxed part is the initiation codon.

[0048] 2. Extraction of coccidia total RNA:

[0049] Take 0.1 g (about 107) of pure sporulated oocysts of E. tenella, put them in a glass homogenizer, add 1 ml of Trizol reagent, grind for 5 minutes to rupture the oocyst wall, and extract the total sporozoite RNA in one step. The specific steps are as follows: put the homogenized solution into a 1.5ml eppendorf tube, add 0.2mL of 24:1 chloroform / isoamyl alcohol mixture, shake the sample vigorously for 15s, put it on ice for 5min, centrifuge at 1200...

Embodiment 2

[0074] Example 2. Preparation of chIL-2 gene:

[0075] 1. Synthesize primers P3 and P4, the sequences are:

[0076] P3: 5'-CTA GAATTC CCTACCCTCGAT TGCAAAGTACTGATCT-3'

[0077] P4: 5'-TTA GTC GAC TTGCAGATATCTCCAAAAGTT-3'

[0078] The underlined part is the introduced enzyme cutting site EcoRI and SalI; the square part is the start codon; the italic letter part is the coagulation factor X a The specific hydrolysis sequence encodes a nucleotide sequence (such as the nucleotide sequence shown at positions 1465-1482 in SEQ ID NO: 1).

[0079] 2. PCR amplification of chIL-2 gene

[0080] Add the following components to a thin-walled PCR tube for PCR amplification:

[0081] Plasmid pBV220-chIL-2 (50ng / μl) 1.0μl

[0082] 2.5mmol / L dNTP 4.0μl

[0083] P3 (50pmol) 1.0μl

[0084] P4 (50pmol) 1.0μl

[0085] 10×PCR buffer 5.0μl

[0086] 25mmol / L MgCl 2 23.0μl

[0087] Taq DNA polymerase (5U / μl) 0.5μl

[0088] wxya 2 O 34.5 μl

[0089] Total 50.0μl ...

Embodiment 3

[0093] The preparation of embodiment 3.DNA vaccine

[0094] 1. Construction of recombinant plasmid pMD18-T-pEtK2 (see image 3 )

[0095] Get 50 μ l of the PCR product obtained in Example 1, electrophoresis on 1% agarose gel, cut out the agarose gel at the band of interest under ultraviolet light, reclaim and purify the fragment of interest with the gel recovery kit of Dalian Bao Biological Company, method Refer to the instruction manual. Take the purified PCR product and connect it with the pMD18-T vector linearized after digestion with BamHI and EcoRI. The reaction system is as follows:

[0096] PCR recovery product 4.0μl

[0097] pMD18-T vector 1.0μl

[0098] Ligation solution I 5.0μl

[0099] Total volume 10.0μl

[0100] The above components were mixed in a thin-walled eppendorf tube and connected overnight at 4°C. The ligation product was transformed into competent Escherichia coli JM109, the plasmid was extracted, and identified by double enzyme digestion and se...

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Abstract

The invention provides an immunoregulation type DNA vaccine with actions of preventing and treating chicken coccidiosis, which comprises calcium regulation bonding domain protein kinase genes pE+K2 of Eimeria tenella and chicken interleukin-2 genes, and a section of proteinase hydrolytic sequence is introduced to the 5' end of the chIL-2 gene.

Description

technical field [0001] The invention relates to the technical field of biological veterinary medicine, and is an immunoregulatory DNA vaccine capable of preventing chicken coccidiosis. This vaccine contains Eimeria tenella (Eimeria tenella) calcium regulation binding domain protein kinase gene pEtK2 and chicken interleukin-2 gene (chIL-2). And a protease-specific hydrolysis sequence was introduced at the 5' end of the chIL-2 gene. This vaccine contains multiple T cell immune response elements. Background technique [0002] Chicken coccidiosis is an important parasitic disease that harms the poultry industry, and the economic loss caused by coccidiosis is about 2 billion pounds worldwide every year. The disease is caused by 7 species of coccidia of the genus Eimeria, among which E. tenella causes the greatest damage. The current drug control of chicken coccidiosis has brought a series of problems. Such as the emergence of drug resistance, the harm caused by drug residues ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/012A61P33/02C12N15/79
Inventor 李祥瑞谢昆严若峰徐立新
Owner NANJING AGRICULTURAL UNIVERSITY
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