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Recombinant protein encoded by Eimeria tenella surface antigen gene SAG6, and applications thereof

A surface antigen gene, Eimeria technology, applied in the field of molecular biology and animal vaccines, can solve the problem that there is no ideal chicken coccidiosis drug and vaccine

Pending Publication Date: 2017-11-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the control of coccidiosis mainly depends on anticoccidiosis drugs, but due to the lack of detailed understanding of coccidiosis cells and molecular biology, there is no ideal drug and vaccine for chicken coccidiosis control so far. Vaccine target is of great significance to the prevention and treatment of chicken coccidiosis

Method used

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  • Recombinant protein encoded by Eimeria tenella surface antigen gene SAG6, and applications thereof
  • Recombinant protein encoded by Eimeria tenella surface antigen gene SAG6, and applications thereof
  • Recombinant protein encoded by Eimeria tenella surface antigen gene SAG6, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Extraction of Eimeria tenella total RNA

[0019] 1) will contain about 1 x 10 8 A Eimeria tenella sporulated oocyst suspension was centrifuged at 3000rpm for 5min, and the supernatant was discarded;

[0020] 2) The precipitate was resuspended by adding 1 ml TRIzol, and then grinded for 20 min with 1 ml RNase-free homogenizer until 90% of the sporozoites burst. After grinding, transfer to an RNase-free 1.5ml centrifuge tube and place at room temperature (15-30°C) for 10 minutes to completely separate the nucleic acid-protein complex;

[0021] 3) Add 0.2ml of chloroform to the suspension, shake vigorously for 15sec, and place at room temperature for 5min;

[0022] 4) Centrifuge at 4°C and 12000rpm for 15min, transfer the colorless upper layer to a new 1.5ml centrifuge tube without RNase, discard the middle layer and the lower layer;

[0023] 5) Precipitate the RNA in the aqueous phase with an equal volume of pre-cooled isopropanol, mix well and place at -...

Embodiment 2

[0027] Embodiment 2: Synthesis of Eimeria tenella total cDNA

[0028] Using the RevertAid First Strand cDNASynthesis Kit from Thermo Company, the extracted Eimeria tenella total RNA was used as a template for reverse transcription. The specific steps are as follows:

[0029] ① Prepare the following mixture in Microtube

[0030]

[0031] ② Perform denaturation and annealing reactions under the following conditions on a PCR machine:

[0032] 65℃ 5min ↓ Rapid cooling on ice

[0033] ③ Prepare the following reverse transcription reaction solution in the above-mentioned Microtube tube:

[0034] The reaction solution after the above-mentioned denaturation and annealing

[0035]

[0036] ④ Perform the reverse transcription reaction on the PCR instrument according to the following conditions:

[0037]

[0038] The cDNA obtained by reverse transcription was stored at -20°C for future use.

Embodiment 3

[0039] Example 3: Acquisition of Eimeria tenella surface antigen gene SAG6

[0040] 1) Primer design

[0041] A pair of primers (pET-28a-SAG6-F and pET-28a -SAG6-R) amplifies the Eimeria tenella surface antigen gene SAG6, specifically as follows:

[0042] pET-28a-SAG6-F: 5'-TGACTGGTGGACAGCAAATGACAATCAAGTACACTGCTTC-3' pET-28a-SAG6-R: 5'-TTAGCAGCCGGATCTCATCAGATGGCGGCTGACGCTGACC-3'

[0043] 2) PCR amplification: PCR amplification was performed using Eimeria tenella cDNA as a template, the reaction system was 50 μL, and the PCR reaction system was as follows:

[0044]

[0045] PCR reaction conditions: pre-denaturation at 98°C for 5 min; cycles of denaturation at 98°C for 5 min, annealing at 57°C for 15 sec, and extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10 min.

[0046] 3) Electrophoresis detection and recovery of PCR products: Electrophoresis of PCR products on 1.8% agarose gel, adjusting voltage to 150V, electrophoresis for 15min, photographic...

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Abstract

The present invention discloses a recombinant protein encoded by an Eimeria tenella surface antigen gene SAG6, and belongs to the field of molecular biology and animal vaccines. According to the present invention, the recombinant protein can be expressed in a prokaryotic system, has good reactionogenicity, can provide strong immunoprotection for chicken, can provide excellent effects superior to other SAG genes in the fields of the relative weight gain, the reduction of cecum diseases of infected chicken, and the anticoccidial index, and can provide important significance for the development and research of new drugs and vaccines for chicken coccidiosis.

Description

technical field [0001] The invention belongs to the fields of molecular biology and animal vaccines, and relates to a recombinant protein encoded by the surface antigen gene SAG6 of Eimeria tenella (E. tenella), and also relates to the application of the recombinant protein. Background technique [0002] Chicken coccidiosis is a parasitic disease that is distributed worldwide and seriously endangers the development of the chicken industry. Eimeria tenella is one of the most pathogenic chicken coccidia, which mainly parasitizes in the chicken intestine and causes chicken coccidiosis characterized by intestinal lesions and seriously endangers the poultry industry. develop. At present, the control of coccidiosis mainly depends on anticoccidiosis drugs, but due to the lack of detailed understanding of coccidiosis cells and molecular biology, there is no ideal drug and vaccine for chicken coccidiosis control so far. Vaccine targets are of great significance for the prevention a...

Claims

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Application Information

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IPC IPC(8): C07K14/455C12N15/70A61K39/012A61P33/02
CPCA61K39/012A61K2039/55566C07K14/455C12N15/70
Inventor 方瑞冯汉利王利霞杨新段茜谭理周艳琴贺兰申邦胡敏赵俊龙
Owner HUAZHONG AGRI UNIV
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