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Preparation and application of fusion protein genetically engineered bacterium of coccidium antigen peptide/IL5

A technology of genetically engineered bacteria and fusion proteins, applied in peptide/protein components, carrier-bound antigen/hapten components, medical preparations containing active ingredients, etc., can solve the problem of low protection of coccidiosis vaccines and achieve prevention Effects of coccidia infection and ease of use

Active Publication Date: 2022-07-12
四川宜美康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is: how to provide a fusion protein of food-grade coccidia antigenic peptide / IL5 and secrete and express the genetically engineered bacteria of the fusion protein, It is used to prevent and treat bacterial diarrhea in livestock, and solve the problem of low protection of existing coccidiosis vaccines

Method used

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  • Preparation and application of fusion protein genetically engineered bacterium of coccidium antigen peptide/IL5
  • Preparation and application of fusion protein genetically engineered bacterium of coccidium antigen peptide/IL5
  • Preparation and application of fusion protein genetically engineered bacterium of coccidium antigen peptide/IL5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The fusion protein of coccidioid antigen peptide / IL5, whose amino acid sequence is shown in SEQ ID No. 1, is obtained by expressing the fusion gene of coccidia antigen peptide / IL5. In this amino acid sequence:

[0046] MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAAVGSSRVDVPG is the signal peptide sequence.

[0047] MRTHLYFLLLTVGISATPLRSNLAELQTWLQQIYQSVDMLNLRIETPVSADDENCIKTLFEGTALLKNNPEMRRFGTFFQSFDKLRPSLTAQLTGEGECDTERKNVKKFIEKLRTFIRKLSRDARV is an IL5 sequence fragment.

[0048]SHGMVMCANAEDKSSVELLQ,GAYMQLSRRGGDAAFDEKEV,GGAGGDGGAGGGTGEEE,NGLSVDETIKVTSAGWTKSE,SSSSCCALLQLEKQDLEQSL,FNDSQRQATKDAGTIAGLNV,ALNDPVVYNDRAVSFVALYN,AWDTSVKEWLVDTGKVYAGG,AWDTSVKEWLVDTGKVYAGG,RLTVRGGLQEKEAVKVTANG,SDALEYWKGGLSQFNDKIPP,GRISSYYTSPLLLSGTCSLS,NPSSSATADCRVVTCTQTNT,KASQRDFQLLKHLTRWYNHM,AEVAAACATKASAEAALLTG为抗原基因蛋白预测片段,通过GSG连接,具有较高的免疫原性。

[0049] FYPSYHSTPQRP is a dendritic cell-inducing peptide that helps antigens target dendritic cells and increases the possibility of antigens being phagocytosed by immune c...

Embodiment 2

[0051] Example 2 Synthesis of fusion gene encoding coccidioid antigen peptide / IL5 fusion protein

[0052] The nucleotide sequence of the fusion gene encoding the fusion protein of Coccidia antigen peptide / IL5 is shown in SEQ ID No.2.

[0053] When a eukaryotic gene is cloned and expressed in prokaryotes, the codons preferred by eukaryotes need to be changed to those preferred by prokaryotes (Bacillus subtilis) to achieve high-efficiency expression. The present invention performs codon optimization on the gene sequence according to the codon preference of the corresponding cells, and designs the nucleotide sequence SEQ ID No.2.

[0054] The fusion gene in this example uses deoxyribonucleotides as substrates, and nucleotide fragments of the target sequence are synthesized on a DNA synthesizer.

Embodiment 3

[0055] Example 3 Construction of Coccidia Antigen Peptide / IL5 Fusion Protein Coding Gene Expression Vector

[0056] In order to connect with the plasmid vector, the homology arm sequence and restriction enzyme site were designed on the upstream and downstream of the coding sequence. That is, GCGGTACCGAGCTCGCTCGAG is added upstream of the nucleotide sequence described in SEQ ID No. 2, and TGACTCGAGGGGCTAGCCGCTGCA is added downstream. The improved nucleotide sequence is shown in SEQ ID No.3. Nucleotide fragments of the target sequence are synthesized on a DNA synthesizer.

[0057] 1) Take the DNA fragment shown in SEQ ID No.3 as a template, take SEQ ID No.5

[0058] (GCGGTACCGAGCTCGCTCGAG) and SEQ ID No. 6

[0059] The nucleotide sequence shown in (TGCAGCGGCTAGCCCCTCGAGTCA) is a primer, and PCR amplification is performed to obtain an amplified fragment of the fusion gene;

[0060] 2) Use XholI enzyme to digest the vector plasmid plasmid 2021 and the amplified fragment, and u...

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Abstract

The invention discloses preparation and application of a fusion protein genetically engineered bacterium of coccidium antigen peptide / IL5. The amino acid sequence of the fusion protein is shown as SEQ ID No. 1. The sequence of the coding gene of the gene is shown as SEQ ID No.2. The protein provided by the invention has remarkable prevention, treatment and regulation effects on avian coccidiosis. The cationic polypeptide compound expressed by the food-grade bacillus subtilis causes acquired immune response of animal intestinal tracts through mucosal immunity, and an anti-coccidiosis antibody is generated. The interleukin 5 is an eosinophilic granulocyte activating factor and can activate eosinophilic granulocytes in intestinal tracts, secrete anti-parasitic active substances and repair intestinal mucosa damage caused by coccidium infection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a food-grade Bacillus subtilis (without drug resistance genes) coccidia antigen peptide / IL5 for preventing and treating avian coccidiosis and a Bacillus subtilis secreted peptide fusion protein, fusion gene, Expression vector and its genetically engineered bacteria. Background technique [0002] Coccidiosis causes billions of losses to livestock and poultry breeding in my country every year. At present, antibiotics are mainly used to suppress coccidiosis outbreaks, but they cannot solve the fundamental problem. The use of antibiotics has resulted in increased drug resistance and higher farming costs. Residues of veterinary drugs endanger human health. [0003] In order to deal with the drug resistance of protozoan parasites such as coccidia, a new drug must be introduced every 1 to 2 years in the world. With the continuous improvement of people's requir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/75C12N15/66C12N1/21A61K39/012A61K39/385A61K38/20A61P33/02A61P1/12C12R1/125
CPCC07K14/455C07K14/5409C12N15/75A61K39/012A61K39/385A61P33/02A61P1/12C07K2319/00A61K38/00A61K2039/552A61K2039/6031Y02A40/70
Inventor 不公告发明人
Owner 四川宜美康科技有限公司
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