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Eimeria tenella gametophyte antigen gam59 gene and application thereof

A technology of Eimeria and antigen genes, applied in application, gene therapy, genetic engineering, etc., can solve the problem of no nucleotide sequence reporting

Inactive Publication Date: 2015-05-06
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, foreign literature has also confirmed that there are two homologues of the Eimeria maxima gametocyte Emgam56 gene in Eimeria tenella, which are Etgam56 and Etgam59, but their nucleotide sequences have not been reported yet.

Method used

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  • Eimeria tenella gametophyte antigen gam59 gene and application thereof
  • Eimeria tenella gametophyte antigen gam59 gene and application thereof
  • Eimeria tenella gametophyte antigen gam59 gene and application thereof

Examples

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Effect test

Embodiment 1

[0044] Example 1: Isolation of the Etgam59 gene from the Eimeria tenella gametophyte gene

[0045] The gametocytes of Eimeria tenella were isolated and purified by conventional methods, and the total RNA was extracted with an RNA extraction kit.

[0046] RT-PCR catch Etgam59 gene:

[0047] Referring to TaKaRa company (TaKaRa RNA LA PCR TM The method provided by Kit (AMV) Ver.1.1): the two-step RT-PCR kit amplifies the Etgam59 gene.

[0048] The Etgam59 gene was captured using an upstream primer (5'-CCCCGGGAACATGACTCGTCTCGCCGCC-3') and a downstream primer (5'-CGAATTCTTACTCAAATCCAAAAGAAGG-3').

[0049] The first step: reverse transcription reaction, the total system is 10μl: 0.5μl total RNA (≤500ng total RNA), 25mM MgCl 2 2μl, 10×RNA PCR Buffer 1μl, RNase Free dH 2 O 4.25 μl, dNTP 1 μl at a concentration of 10 mM, RNase Inhibitor 0.25 μl, 5 μ / μl AMV reverse transcriptase 0.5 μl and Oligo dT linker primer 0.5 μl. Reaction steps: reverse transcription at 42°C for 30 minutes; ...

Embodiment 2

[0051] Embodiment 2: Amplification of expression fragment Etgam59

[0052] According to the ORF sequence of the Eimeria tenella Etgam59 gene and the restriction site of the plasmid pET28a (purchased from Invitrogen), after removing the signal peptide, specific primers were designed to amplify the gene fragment for expression.

[0053] According to the enzyme cutting sites on the plasmid pET28a, after sequence analysis of the Etgam59 gene, the signal peptide was eliminated, EcoRI and HindIII enzyme cutting sites were selected, and the specific primers were designed as: upstream primer (5'-CCGGAATTCATGCCCACGGCCATCCCC- 3') contains an EcoRI restriction site and 3 protective bases, and the downstream primer (5'-CCGAAGCTTTTACTCAAATCCAAAAGA-3') contains a HindⅢ restriction site and 3 protective bases.

[0054] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; 2 μl of upstream and downstream primers with a concentration of 10 μM, 8 μl of dNTP (2.5 mM); 30.5 μl...

Embodiment 3

[0055] Embodiment 3: the construction of prokaryotic expression vector

[0056] Double digestion of pET28a:

[0057] EcoRI and HindⅢ are Fermentas products. 100 μl digestion system: 10 μl 10× FastDigest Green Buffer, 50 μl vector pET28a, 5 μl each of FastDigest EcoRI and FastDigest HindIII, 30 μl sterilized ddH 2 O. React at 37°C for 12 hours, perform 1.2% agarose gel electrophoresis, and recover the product from the gel.

[0058] The PCR product purified in double enzyme digestion embodiment 2:

[0059] EcoRI and HindⅢ are Fermentas products. 100 μl digestion system: 10 μl 10× FastDigest Green Buffer; 20 μl purified PCR (Etgam59) product; 5 μl each of FastDigest EcoRI and FastDigest HindIII; 60 μl sterilized ddH 2 O. React at 37°C for 12 hours, perform 1.2% agarose gel electrophoresis, and recover the digested product.

[0060] Link reaction:

[0061] Solution Ⅰ is a TaKaRa product. 9 μl double-digested vector pET28a, 1 μl double-digested PCR product, 10 μl solution ...

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Abstract

The invention belongs to the field of gene engineering vaccines, and concretely relates to an eimeria tenella gametophyte antigen gene, expression and application of an expression product. The size of the open reading frame of the gene is 1617 bp, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. A prokaryotic expression vector pET28a-Etgam59 is constructed based on the gene, and is transformed into BL21 host bacterium for inducible expression. The expression product can be recognized by chicken convalescent serum, which shows that recombinant protein possesses immunogenicity. The obtained serum of an immunized mouse of a recombinant eukaryotic expression plasmid pcDNA3.1-Etgam59 constructed by amplifying the gene is capable of specifically combined with the expression product of pET28a-Etgam59, which shows that the gene is applicable to development of a chicken coccidiosis gene engineering vaccine.

Description

technical field [0001] The invention relates to a gametocyte antigen gene of Eimeria tenella, a polypeptide encoded by the gene, a carrier containing the gene, an acquisition method of the gene, an in vitro expression method of the polypeptide and functional identification. Specifically, the gene of the present invention comes from the gametophyte of the sexual reproduction stage of Eimeria tenella. Background technique [0002] Chicken coccidiosis is a protozoan disease that seriously endangers the chicken industry caused by one or several coccidia of the genus Eimeria parasitizing in the small intestine or cecum mucosa of chickens. It is estimated that the annual global loss due to coccidiosis is 3 billion Dollar. The annual consumption of anticoccidiostats alone in my country is about 600-1.8 billion yuan, and the direct and indirect economic losses caused by chicken coccidiosis are difficult to estimate. For a long time, the prevention and treatment of chicken coccidio...

Claims

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Application Information

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IPC IPC(8): C12N15/30C07K14/455C12N15/70A61K48/00A61P33/02
Inventor 陶建平朱玉兰刘丹丹许金俊
Owner YANGZHOU UNIV
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