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Immunoloregulation type DNA vaccine capable of preventing Eimeria necatrix

A DNA vaccine and Eimeria technology, applied in the field of DNA vaccines, can solve the problems of high cost, existence of scattered poison, difficult preservation, etc., and achieve the effects of long maintenance time, high safety and good thermal stability

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

The successful development and application of coccidiosis live capsule vaccine provides a technical means that can replace drugs to control chicken coccidiosis, but live vaccines inevitably have disadvantages such as high cost, difficulty in storage, loose virus and potential disease-causing threats

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Preparation of embodiment 1.NA4 gene

[0048] 1.1 Synthetic primers

[0049] According to the nucleotide sequence of the sporozoite surface antigen TA4 gene of Eimeria tenella, NA4 primers P1 and P2 were designed using the software primerpremier5.0, and sent to TaKaRa to synthesize primers. The sequence is as follows:

[0050] P1: 5'—G GGATCC GCTCGTCTCCTCT-3',

[0051] P2: 5'— GAATTCC TAGAAGAGAGCGAAA—3'.

[0052] The underlined parts are the introduced restriction sites BamHI and EcoRI, respectively; the boxed part is the initiation codon.

[0053] 1.2 Extraction of total RNA from sporozoites of poisonous Eimeria

[0054] A one-step method was used to extract the total RNA of sporulated oocysts, and the specific operation steps were as follows: Take about 10 sporulated oocysts of purified E.necatrix 7 Each was placed in a nuclease-free glass homogenizer treated with DEPC, and 1ml Trizol reagent was added, and rapidly ground for 5 minutes. Put the homogenized...

Embodiment 2

[0065] The preparation of embodiment 2.DNA vaccine

[0066] 2.1 Construction of recombinant plasmid pVAX-NA4 (see Figure 4 )

[0067] The cloned plasmid vectors pMD18-T-NA4 and pVAX1 were digested with BamHI and EcoRI respectively, and the NA4 target gene and pVAX1 large fragment were recovered, ligated according to an appropriate ratio, and the ligated product was transformed into competent Escherichia coli BL21, and the plasmid was extracted with BamHI and EcoRI It was identified as pVAX-NA4 by double enzyme digestion.

[0068] 2.2 Construction of recombinant plasmid pVAX-NA4-IL-2 (see Figure 5-8 )

[0069] 2.2.1 Synthetic primers

[0070] In order to construct the recombinant plasmid of tandem cytokines, use the software primer premier5.0 to design NA4 specific primer P3 without stop codon, and add coagulation factor X at the 3' end of NA4 a The specific hydrolysis sequence encodes the nucleotide sequence (box part), and the underlined part is the introduced enzyme cu...

Embodiment 3

[0075] The detection of embodiment 3.DNA vaccine expression situation in chicken body

[0076] The pVAX-NA4 and pVAX-NA4-IL-2 recombinant plasmids were extracted respectively, and 14-day-old chicks (100 μg / piece) were injected intramuscularly into the chest, and the injected site (breast muscle) and non-injected muscle (leg) were collected 7 days later.

[0077] 3.1 RT-PCR detection of DNA vaccine transcription

[0078] 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 ).

[0079] 3.2 Detection of translation of DNA vaccine by Western blot

[0080] 3.2.1 Preparation of recombinant pET28a-NA4 protein antiserum

[0081] Transform pET28a-NA4 into Escherichia coli BL21 for prokaryotic expression. When the OD600 of the bacteria is 0.5, induce with isopropyl-β-D-thiogalactosi...

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Abstract

The invention relates to a chicken toxic Eimeria acervulina immunoregulation type DNA vaccine, which belongs to the technical field of biological veterinary medicines. The molecular biology technology is used for connecting toxic Eimeria sporozoite surface antigen NA4 genes and chIL-2 genes in series to construct a fusion expressing carrier pVAX-LDH-IL-2. The vaccine comprises the inserted toxic Eimeria sporozoite surface antigen NA4 genes and the chIL-2 genes. The vaccine comprises a plurality of T cell immune response elements, and has the advantages of high safety, long keeping time in bodies, simple preparation, good heat stability, convenient storage and transportation, time saving and labor saving when in use and the like, thus preventing and curing chicken coccidiosis effectively. Tests prove that the immunoregulation type DNA vaccine pVAX-NA4-IL-2 has good immune protection effect in coccidian resistant infections.

Description

technical field [0001] The invention relates to an immunoregulatory DNA vaccine capable of preventing Eimeria coccidiosis caused by chicken poisoning, and belongs to the technical field of biological veterinary medicine. The vaccine contains the sporozoite surface antigen NA4 gene of Eimeria necatrix and chicken interleukin-2 gene (chIL-2). This vaccine contains multiple T cell immune response elements. Background technique [0002] Chicken coccidiosis is a protozoan disease caused by Eimeria coccidiosis. The pathogens are poisonous Eimeria (E.necatrix), Eimeria tenella (E.tenella), and pile-type Eimeria. E. necatrix, E. maxima, E. branetti, E. praecox, E. placid (E. mitis). Among them, poisonous Eimeria coccidiosis is highly pathogenic and is one of the most serious diseases in industrialized chicken farming. It can cause digestive dysfunction in chickens, stunted growth, slowed weight gain, and decreased egg production rate. Since the 1940s, countries have mainly used ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/012A61K48/00A61P33/02
Inventor 李祥瑞严若峰徐立新宋小凯张娜
Owner NANJING AGRICULTURAL UNIVERSITY
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