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Eimeria tenella rhoptry protein 41 as well as preparation method and application thereof

A technology of Eimeria and Eimeria, applied in the direction of immunoglobulin, botany equipment and method, biochemical equipment and method, etc., can solve the problem that there is no research report on Eimeria coccidioid protein, etc. question

Active Publication Date: 2020-05-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no research report on the Eimeria rhizome protein

Method used

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  • Eimeria tenella rhoptry protein 41 as well as preparation method and application thereof
  • Eimeria tenella rhoptry protein 41 as well as preparation method and application thereof
  • Eimeria tenella rhoptry protein 41 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Preparation of EtROP41 recombinant protein

[0043] The accession number of the EtROP41 protein of Eimeria tenella described in the present invention in the ToxoDB database is ETH_00005405 (https: / / toxodb.org / toxo / app / record / gene / ETH_00005405), the gene encoding the protein The length of the fragment sequence is 1527bp.

[0044] 1. Cloning of EtROP41 gene and construction of recombinant plasmid

[0045] Design primers to amplify the EtROP41 gene, and the primers are shown in the table below:

[0046] Table 1

[0047]

[0048] EtROP41 was amplified using the cDNA of Eimeria tenella sporozoites as a template, and the reaction system was as follows:

[0049] Table 2

[0050]

[0051] 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 for agarose gel...

Embodiment 2

[0057] Example 2: Identification of EtROP41 antigenicity

[0058] 1. Preparation of Polyclonal Antibody

[0059] After EtROP41-His recombinant protein was emulsified with the same volume of Freund's complete adjuvant, BALB / c mice were immunized at a dose of 100 μg / mouse for the first time; with recombinant protein and Freund's incomplete adjuvant emulsified, BALB / c mice were immunized with a dose of 50 μg / mouse Mice; then emulsified with Freund's complete adjuvant, immunized BALB / c mice at a dose of 50 μg / only, and carried out the second and third immunizations, with an interval of 14 days between each immunization, and 10 days after the second and third immunizations, respectively. Collect blood and separate serum from the orbit of the day, and use ELISA to detect the antibody titer. When the titer reaches 10 6 As above, mouse eyeballs were removed, blood was collected, serum was separated, and mouse-derived EtROP41 polyantiserum was prepared.

[0060] 2. Preparation of chi...

example 4

[0102] Example 4: Comparison of the immune protection effect of EtROP41 recombinant protein and other recombinant proteins

[0103] In order to compare the immune protection effect of EtROP41 recombinant protein and other recombinant proteins, three surface proteins EtSAG2 of Eimeria tenella were selected (ToxoDB sequence number: ETH_00034890; https: / / toxodb.org / toxo / app / record / gene / ETH_00034890), EtSAG16 (ToxoDB serial number: ETH_00013140; https: / / toxodb.org / toxo / app / record / gene / ETH_00034890), EtSAG (ToxoDB serial number: ETH_00008670; https: / / toxodb.org / toxo / app / record / gene / ETH_00008670). Express and purify EtSAG, EtSAG2 and EtSAG16 recombinant proteins by the same method as that used to prepare EtROP41 recombinant proteins, see Figure 4 , the three recombinant proteins were carried out by the method described in Example 3 to evaluate the immune protection effect, and the results of various detection indicators are shown in Table 9. The results showed that the protecti...

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Abstract

The invention discloses an Eimeria tenella rhoptry protein 41 as well as a preparation method and application thereof. The protein is a new-found protein capable of highly expressing in a sporozoite stage of Eimeria tenella and is a protein relevant with invasion of sporozoites in host cells, an amino acid sequence of the protein includes an amino acid sequence represented by SEQ ID NO.1. An EtROP41 gene is linked with an escherichia coli expression vector to form a recombinant expression vector, and a recombinant EtROP41 protein is expressed in escherichia coli, is purified and is mixed withan adjuvant so as to form a chicken Eimeria tenella ROP41 recombinant protein vaccine; and by immunizing chicks through the vaccine, the infection of the Eimeria tenella can be effectively resisted, the survival rate of the chicks can be increased, the relative weight gain rate of the chicks can be increased, and the discharge rate of oocysts of the Eimeria tenella can be decreased, so that the vaccine is a novel recombinant protein vaccine with a good immune protection effect and has the potential as a vaccine researched and developed for preventing a chicken Eimeria tenella disease.

Description

technical field [0001] The invention relates to the technical field of biological veterinary medicine, in particular to a coccidium tenella rod protein 41 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. It is distributed worldwide. The incidence of chicken coccidiosis is as high as 50-70%. The mortality rate is 20-30%, up to 80% in severe cases, bringing huge economic losses to the poultry industry. The U.S. Department of Agriculture lists the disease as one of the five most serious diseases for poultry. There are 7 kinds of chicken coccidia recognized in the world, among which Eimeria tenella parasitizing in the cecum is the most pathogenic and the most harmful. [0003] At present, the prevention and control of coccidiosis mainly relies on anticoccidial drugs and live vaccines. However, due to...

Claims

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Application Information

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IPC IPC(8): C07K14/455C12N15/30C12N15/11C12N15/70C07K16/20C07K16/06A61K39/012A61P33/02
CPCA61K39/012A61K2039/552A61P33/02C07K14/455C07K16/06C07K16/20C12N15/70
Inventor 刘群宋星桔刘晶杨旭许建海
Owner CHINA AGRI UNIV
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