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Gametophyte antigen gam22 gene of Eimeria necatrix and application of gene

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

Inactive Publication Date: 2013-08-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are no reports of related gene sequences and their recombinant expression at home and abroad.

Method used

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  • Gametophyte antigen gam22 gene of Eimeria necatrix and application of gene
  • Gametophyte antigen gam22 gene of Eimeria necatrix and application of gene
  • Gametophyte antigen gam22 gene of Eimeria necatrix and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Isolation of the gam22 gene from the Eimeria gametocyte gene

[0046] Conventional methods were used to separate and purify the gametocytes of Eimeria venomosa, and the total RNA was extracted with an RNA extraction kit.

[0047] RT-PCR catch gam22 gene:

[0048] Refer to TaKaRa company (TaKaRa RNA LA PCR TM The method provided by Kit (AMV) Ver.1.1): two-step RT-PCR kit to amplify the gam22 gene.

[0049] The Engam22 gene was captured using an upstream primer (5'-ACCCCAAAATAAAATCAAAGGC-3') and a downstream primer (5'-CCATGAAGATCTCAGACGTAGC-3').

[0050] 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 Buffer1μl, RNase Free dH 2 O4.25μl, concentration of 10mM dNTP1μl, RNase Inhibitor0.25μl, 5u / μl AMV reverse transcriptase 0.5μl and Oligo dT linker primer 0.5μl. Reaction steps: reverse transcription at 42°C for 30 minutes; denaturation at 99°C for 5 minutes; cooling at...

Embodiment 2

[0052] Embodiment 2: the amplification of expression fragment Engam22

[0053] According to the ORF sequence of the Eimeria virulence gam22 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.

[0054] According to the enzyme cutting sites on the plasmid pET28a, after sequence analysis of the Engam22 gene, the signal peptide was eliminated, EcoR I and Hind III enzyme cutting sites were selected, and the specific primers were designed as: upstream primer (5'-TCGGAATTCGACGGAGCACCTGAG -3') contains an EcoR I restriction site and 3 protective bases, and the downstream primer (5'-GCGAAGCTTTTAGTTGATGTCGGT-3') contains a HindⅢ restriction site and 3 protective bases.

[0055] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; 1 μl of upstream and downstream primers with a concentration of 10 μM, 4 μl of dNTP (10 mM); 35 μl o...

Embodiment 3

[0056] Embodiment 3: the construction of prokaryotic expression vector

[0057] Double digestion of pET28a:

[0058] EcoRI and HindⅢ are Fermentas products. The 50 μl digestion system is: 5 μl 10× FastDigest Green Buffer, 20 μl vector pET28a, 2.5 μl each of FastDigest EcoRI and FastDigest HindⅢ, 20 μl ddH 2 O. React at 37°C for 2 hours, electrophoresis on a 1.2% agarose gel, and recover the product from the gel.

[0059] The PCR product purified in double enzyme digestion embodiment 2:

[0060] EcoRI and HindⅢ are Fermentas products. 100 μl enzyme digestion system: 10 μl 10× FastDigest Green Buffer; 20 μl purified PCR (Engam22) product; 5 μl each of FastDigest EcoRI and FastDigest HindⅢ; 60 μl ddH 2 O. React at 37°C for 2 hours, electrophoresis on a 1.2% agarose gel, and recover the digested product.

[0061] Link reaction:

[0062] Solution Ⅰ is a TaKaRa product. 8 μl double-enzyme-cut vector pET28a, 2 μl double-enzyme-cut PCR product, 10 μl solution Ⅰ, ligated overn...

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Abstract

The invention belongs to the field of genetic engineering vaccines, and in particular relates to a gametophyte antigen gene of chicken Eimeria necatrix, expression and application of an expression product of the gene. An open reading frame of the gene has the size of 561 bp. The gene is constructed into a prokaryotic expression vector pET28a-Engam22 to transform BL21 host bacteria to perform inducible expression. The expression product can be identified by chicken rehabilitation serum, and the fact shows that recombinant protein has immunogenicity. The recombinant eukaryotic expression plasmid pcDNA3.1-Engam22 constructed by the gene is used for immunizing chicks and can induce to generate humoral immunity and cellular immunity, and the fact shows that the gene can be applied to development of a chicken coccidiosis gene engineering vaccine.

Description

technical field [0001] The invention relates to a gametocyte antigen gene of Eimeria necatrix, a polypeptide coded 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 gallisepticum. 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 coc...

Claims

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Application Information

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IPC IPC(8): C12N15/30C07K14/455C12N15/70A61K48/00A61P33/02
Inventor 陶建平刘丹丹曹李琴许金俊
Owner YANGZHOU UNIV
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