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Eimeria tenella microneme protein-2 mutant EtMIC2-1130 of chickens

A technology of Eimeria and mutants, applied in the field of genetic engineering and genetic engineering, can solve the problems of high production cost, inability to fundamentally improve antigen immunogenicity, safety of live vaccines, etc.

Inactive Publication Date: 2016-10-26
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccines on the market are mainly a mixture of live oocysts of various Eimeria gallinarum, which have a good immune protection effect, but due to safety problems and high production costs of live vaccines, researchers will spend more energy For the development of new molecular vaccines based on subunits
[0003] The development of new molecular vaccines relies on antigens with good antigenicity and immunogenicity. Due to the complex antigenic structure and life history of coccidia, the difficulty of antigen identification and the limitation of antigen immunogenicity screening hinder the development of subunit vaccines.
Researchers have also carried out a lot of research to improve the immune protection effect of antigens, including the development of new adjuvants, non-specific immunity induced by probiotics, etc., but none of these can fundamentally improve the immunogenicity of antigens

Method used

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  • Eimeria tenella microneme protein-2 mutant EtMIC2-1130 of chickens
  • Eimeria tenella microneme protein-2 mutant EtMIC2-1130 of chickens
  • Eimeria tenella microneme protein-2 mutant EtMIC2-1130 of chickens

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The acquisition of embodiment 1 mutant EtMIC2-1130

[0035] First, according to the EtMIC2 nucleotide sequence published by GenBank (GenBank: KC333870.1) artificially synthesized natural EtMIC2 sequence, the obtained EtMIC2 nucleotide sequence, its nucleotide sequence is shown in Seq ID No: 3, and then it was compared with pEASY-T1 vector ligation modeling EtMIC2-T1;

[0036]Then use EtMIC2-T1 as a template, use EtMIC2-951-F and EtMIC2-951-R as primers, and use Diversify PCR Random Mutagenes Kit (Clontech, USA) to perform error-prone PCR amplification of EtMIC2 mutants to obtain error-prone PCR products; The primers used are shown in Table 1:

[0037] Table 1. The mutant EtMIC2-1130 specific primers

[0038]

Embodiment 2

[0039] The preparation of mutant described in embodiment 2

[0040] The error-prone PCR product obtained in Example 1 was connected to the pEASY-T1 vector without purification, and then transferred into Top 10 competent cells, coated with ampicillin LB agar plate, and cultivated in a 37°C incubator until single Clonal colonies increased to 10 7 So far, construct the EtMIC2 gene mutation library;

[0041] The above-mentioned recombinant plasmid containing the gene mutation library and the pCTCON2 plasmid were double-digested with Nhe I and Sal I, and the double-digested gene mutation library was connected to the pCTCON2 vector and then transfected into Saccharomyces cerevisiae EBY100 (purchased from Invitrogen) to obtain a recombinant Strain EBY100 / EtMIC2, positive colonies identified by PCR (primers used for design are shown in Table 2); take the strain EBY100 containing the recombinant plasmid, spread it on the SD-CAA solid medium in a 30°C incubator, and cultivate it for 48...

Embodiment 3

[0049] Example 3 Determination of the immune protection effect of mutant EtMIC2-1130

[0050] The 1-day-old chicks were weighed, and the chickens with large weight differences were removed. A total of 100 chicks were divided into 4 groups, namely PBS-I (non-immune and non-infection group), PBS-II (non-immune infection group), natural protein EtMIC2, mutant EtMIC2 immunization group, 25 chicks in each group. Breed in hood. At the age of 7 days, chickens in each group were subcutaneously immunized with a final concentration of 0.5 mg / mL of protein or PBS 100 μL in Freund's complete adjuvant emulsification, and at the age of 14 days, they were boosted with Freund's incomplete adjuvant Emulsify 100 μL of protein or PBS corresponding to a final concentration of 0.5 mg / mL. The 21-day-old chicks were orally inoculated with 30,000 sporulated oocysts except the PBS-I group. The immune protection effect of the mutant protein was determined based on the body weight growth of immunized...

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Abstract

The invention relates to the field of gene engineering and provides an eimeria tenella microneme protein-2 mutant EtMIC2-1130 of chickens. The mutant is obtained as follows: in-vitro orthogenesis is performed with EtMIC2 (GenBank:KC333870.1) of wild eimeria tenella SD-01 as basic protein, a gene mutation library is established, yeast is used for surface display of a protein mutant library, altered protein with immunogenicity and immune protection remarkably improved is screened by means of sorting by a flow cytometry, animal immune protection effect evaluation and the like, and the mutant is obtained finally. The mutant altered protein has six amino acid mutation sites; the immune protection effect ACI of altered EtMIC2 is improved by about 21.94% while compared with that of natural EtMIC2, and the mutant can serve as a vaccine to be applied in the field of resistance to infection by eimeria tenella of the chickens.

Description

technical field [0001] The invention relates to the field of genetic engineering and genetic engineering, and provides an Eimeria tenenae egg-2 mutant EtMIC2-1130. Background technique [0002] Chicken coccidiosis is a type of intestinal protozoan disease caused by obligate intracellular parasitic Eimeria. It is estimated that the cost of preventing and treating coccidiosis in the world will exceed 3 billion U.S. dollars every year, and China will spend more than 3 billion yuan every year. At present, the prevention and control of coccidiosis mainly relies on drugs and vaccines. With the emergence of drug-resistant strains and legislative restrictions on anticoccidiosis drugs, people pay more attention to immune prevention. Vaccines on the market are mainly a mixture of live oocysts of various Eimeria gallinarum, which have a good immune protection effect, but due to safety problems and high production costs of live vaccines, researchers will spend more energy For the deve...

Claims

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Application Information

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IPC IPC(8): C07K14/455A61K39/012A61P33/02
CPCA61K39/012A61K2039/552A61K2039/55566C07K14/455
Inventor 赵孝民陈正涛韩林臻李宏梅王方昆
Owner SHANDONG AGRICULTURAL UNIVERSITY
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