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Eimeria tenella microcorticoid protein 2 related protein, and preparation method and application thereof

A technology of Eimeria and micronemetein, applied in immunoglobulin, chemical instruments and methods, veterinary vaccines, etc., can solve the problems of limited protective effect, limited expression, and low immunogenicity of recombinant proteins , to achieve good immune protection effect, large expression, easy expression and purification

Inactive Publication Date: 2020-05-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, a number of researchers reported that a series of recombinant proteins such as TA4, SO7, 3-1E, SAG, HSP70, MIC1, MIC2 and 5401 of Eimeria tenella were used as immune antigens, with different degrees of immune protection. effect, but the protective effect of most recombinant proteins is limited, mainly due to their low immunogenicity or limited expression during the parasite life cycle
Existing studies have shown that although different antigens can induce host immune responses to a certain extent and have certain anti-coccidiosis effects, they still have certain limitations as vaccines for preventing coccidiosis in chickens, so there is no commercial chicken balls yet. The application of recombinant protein vaccines against coccidiosis in production requires further screening of effective immune protection antigens to lay the foundation for the development of recombinant protein vaccines against chicken coccidiosis

Method used

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  • Eimeria tenella microcorticoid protein 2 related protein, and preparation method and application thereof
  • Eimeria tenella microcorticoid protein 2 related protein, and preparation method and application thereof
  • Eimeria tenella microcorticoid protein 2 related protein, and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of EtM2AP recombinant protein

[0042] In the present invention, the protein of Eimeria tenella is EtM2AP (the accession number in the ToxoDB database is ETH_00006930 (https: / / toxodb.org / toxo / app / record / gene / ETH_00006930), the length of the gene fragment encoding the protein It is 969bp.

[0043] 1. Cloning of EtM2AP gene and construction of recombinant plasmid

[0044] The primer sequences for amplifying the EtM2AP gene are shown in Table 1:

[0045] Table 1

[0046]

[0047] Using the cDNA of Eimeria tenella sporozoites as a template, EtM2AP was amplified, and the reaction system was as follows:

[0048] Table 2

[0049]

[0050] PCR reaction conditions: pre-denaturation at 94°C for 10 min, followed by 30 cycles, including denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 90 s, and final extension at 72°C for 10 min. The PCR product was sampled and tested by agarose gel electrophoresis to identify...

Embodiment 2

[0055] Example 2: Identification of EtM2AP antigenicity

[0056] 1. Preparation of Polyclonal Antibody

[0057] After mixing and emulsifying EtM2AP-His recombinant protein with the same volume of Freund's complete adjuvant, immunize BALB / c mice with 100 μg of recombinant protein per mouse for the first time; mixing and emulsifying recombinant protein with Freund's incomplete adjuvant at 50 μg per mouse BALB / c mice were immunized for the second and third times, with an interval of 2 weeks between each immunization. 10 days after the second and third immunizations, the blood was collected from the orbit, the serum was separated, and the antibody titer was detected. When the titer reached 10 6 After the above, the mice were removed from the eyes and blood was collected to separate the serum, and the mouse EtM2AP polyantiserum was prepared.

[0058] 2. Preparation of chicken Eimeria tenella positive serum

[0059] Orally infect 3-week-old SPF chickens with 2000 sporulated oocys...

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Abstract

The invention discloses an Eimeria tenella microcorticoid protein 2 related protein, and a preparation method and an application thereof. The protein is a newly discovered protein which is highly expressed by Eimeria tenella in each developmental stage. The amino acid sequence of the protein comprises an amino acid sequence represented by SEQ ID NO. 1. An EtM2AP gene is inserted into an Escherichia coli expression vector pET28a to obtain a recombinant expression vector, the recombinant expression vector is transferred into Escherichia coli to express a chicken Eimeria tenella recombinant protein (EtM2AP), and the recombinant protein is purified, and then is mixed with an adjuvant to prepare an EtM2AP recombinant protein vaccine. The vaccine can effectively increase the survival rate and the relative weight gain rate of chicks and reduce the coccidiosis oocyst discharge amount of the immunized chicks, and the EtM2AP recombinant protein vaccine has a good immune protection effect and hasthe potential of being used as a vaccine for preventing chicken Eimeria tenella coccidiosis.

Description

technical field [0001] The invention relates to the technical field of biological veterinary medicine, in particular to a micronematin 2-related protein of Eimeria tenella and its preparation method and application. Background technique [0002] Chicken coccidiosis is a parasitic protozoan disease mainly caused by intestinal lesions. It is one of the most common diseases in intensive chicken farms and is distributed worldwide. The incidence of chicken coccidiosis is as high as 50% to 70%, the mortality rate is 20% to 30%, and it is as high as 80% in severe cases. Chicken coccidiosis is one of the diseases that must be prevented in the chicken industry. Huge economic losses, mainly including the cost of prevention and control and the loss of production performance (such as low feed conversion rate, slow growth, death), the US Department of Agriculture listed the disease as one of the five most serious diseases for poultry. There are 7 kinds of chicken coccidia recognized in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/455C12N15/11C12N15/70C07K16/20A61K39/012A61P33/02
CPCA61K39/012A61K2039/552A61P33/02C07K14/455C07K16/20C12N15/70
Inventor 刘群宋星桔刘晶杨旭许建海
Owner CHINA AGRI UNIV
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