Immunity regulating type DNA vaccine for preventing and treating chicken coccidiosis

A DNA vaccine and immune regulation technology, applied in the field of DNA vaccines, can solve the problems of not being fully applicable to laying hens and broiler chickens, high cost, and difficult inoculation volume

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of embodiment 1.TA4 gene:

[0041] 1. Synthetic primers P1 and P2:

[0042] The following primers were designed according to the published nucleotide sequence of the TA4 gene:

[0043] P1: 5'- GGATCCG ATGAACAAGCTGA-3′

[0044] P2: 5'- GAATTC AAAGAGAGCGAAAGCGGA-3'

[0045] The underlined parts are the introduced enzyme cutting sites BamHI and EcoR I respectively.

[0046] 2. PCR amplification of TA4 gene

[0047] Add the following components to a thin-walled PCR tube for PCR amplification:

[0048] Plasmid pUC18-TA4 (50ng / μl) 1.0μl

[0049] 2.5mmol / L dNTP 4.0μl

[0050] P1 (50pmol) 1.0μl

[0051] P2 (50pmol) 1.0μl

[0052] 10×PCR buffer 5.0μl

[0053] 25mmol / L MgCl 2 23.0μl

[0054] Taq DNA polymerase (5U / μl) 0.5μl

[0055] wxya 2 O 34.5 μl

[0056] Total 50.0μl

[0057] After centrifuging and mixing the above components, denature at 95°C for 5 minutes on a PCR instrument; 30 cycles at 94°C for 60 seconds, 58°C for 60 sec...

Embodiment 2

[0059] Example 2. Preparation of chIL-2 gene:

[0060] 1. Synthesize primers P3 and P4, the sequences are:

[0061] P3: 5'-CTA GAATTC CCTACCCTCGAT TGCAAAGTACTGATCT-3'

[0062] P4: 5'-TTA GTC GAC TTGCAGATATCTCCAAAAGTT-3'

[0063] The underlined part is the introduced enzyme cutting site EcoR I and SalI; the box 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 649-666 in SEQ ID NO: 1).

[0064] 2. PCR amplification of chIL-2 gene

[0065] Add the following components to a thin-walled PCR tube for PCR amplification:

[0066] Plasmid pBV220-chIL-2 (50ng / μl) 1.0μl

[0067] 2.5mmol / L dNTP 4.0μl

[0068] P3 (50pmol) 1.0μl

[0069] P4 (50pmol) 1.0μl

[0070] 10×PCR buffer 5.0μl

[0071] 25mmol / L MgCl 2 23.0μl

[0072] Taq DNA polymerase (5U / μl) 0.5μl

[0073] wxya 2 O 34.5 μl

[0074] Total 50.0μl

[0...

Embodiment 3

[0078] The construction of embodiment 3.DNA vaccine

[0079] 1. Construction of recombinant plasmid pMD 18-T-TA4 (see image 3 )

[0080]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 to the pMD18-T vector, and the reaction system is as follows:

[0081] PCR recovery product 4.0μl

[0082] pMD18-T vector 1.0μl

[0083] Ligation solution I 5.0μl

[0084] Total volume 10.0μl

[0085] 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, and the plasmid was extracted, identified by double enzyme digestion with BamHI and EcoRI and sequenced, and determined to...

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Abstract

The invention provides an immunoregulation type DNA vaccine with actions of preventing and treating chicken coccidiosis, which comprises sporozoite surface antigen genes TA4 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 belongs to the field of biological products and relates to an immunoregulatory DNA vaccine capable of preventing chicken coccidiosis. The vaccine contains Eimeria tenella sporozoite antigen gene TA4 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 endangers 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 to the environment, and so on. These issues have attracted i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/012A61P33/02
Inventor 李祥瑞徐前明严若峰徐立新
Owner NANJING AGRICULTURAL UNIVERSITY
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