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STa-LTB-STb fusion protein of enterotoxin of escherichia coli and encoding gene and application thereof

A fusion protein and fusion gene technology, applied in the biological field, can solve the problems of not being able to fight against pathogenic factors-enterotoxin, the narrow protection scope of vaccines, etc., and achieve the effects of improving the protection effect, preventing harm, and simplifying the protein purification process.

Inactive Publication Date: 2019-03-15
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] The technical problem mainly solved by the present invention: the existing vaccine against ETEC diarrhea in piglets has a narrow range of protection and can hardly resist enterotoxin, an important pathogenic factor of ETEC. Between this, the present invention utilizes the principle of molecular biology and genetic engineering technology The two subunits STa and STb of the ST enterotoxin were connected to both sides of the carrier protein LTB enterotoxin protein, and the Pichia pastoris eukaryotic expression system was used to express the fusion protein exogenously, which overcomes the previous use of Escherichia coli for prokaryotic expression. The shortcoming of expressing easy formation of inclusion bodies ensures the activity and immunogenicity of ETEC enterotoxins ST and LT

Method used

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  • STa-LTB-STb fusion protein of enterotoxin of escherichia coli and encoding gene and application thereof
  • STa-LTB-STb fusion protein of enterotoxin of escherichia coli and encoding gene and application thereof
  • STa-LTB-STb fusion protein of enterotoxin of escherichia coli and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Construction of STa-LTB-STb fusion gene

[0101] (1), enterotoxin gene LTB PCR amplification

[0102] The PCR reaction template is ETEC F4ac cell lysate (the strain was purchased from the China Veterinary Drug Control Institute, the number is C83902), and the primers, reaction system and reaction conditions used are as follows:

[0103] Primers:

[0104] name

[0105] reaction system:

[0106]

[0107] Reaction conditions: 95°C for 5 min, (94°C for 30 s, 55°C for 30 s, 72°C for 1 min) amplification for 30 cycles, and 72°C for 10 min.

[0108] (2), enterotoxin gene STb PCR amplification

[0109] Primers:

[0110] name

[0111] reaction system:

[0112]

[0113] Reaction conditions: 95°C for 5 min, (94°C for 30 s, 55°C for 30 s, 72°C for 1 min) amplification for 30 cycles, and 72°C for 10 min.

[0114] (3), enterotoxin gene STa PCR amplification

[0115] The size of the STa gene is only 54bp. According to the principle of overlap extension...

Embodiment 2

[0136] Eukaryotic Expression of Enterotoxin STa-LTB-STb Fusion Protein

[0137] (1) Construction of eukaryotic expression vector of enterotoxin STa-LTB-STb fusion gene

[0138] Using the principles and methods of molecular biology, the STa-LTB-STb fusion gene was first connected to the pMD-19T cloning vector (full name: pMD TM -19T Vector Cloning Kit (manufacturer: Takara Company) to obtain the recombinant cloning T vector, and use the heat shock method to introduce the recombinant T vector into E.coli DH5α competent cells. The specific operation is: take 100 μl E.coli DH5α competent cells and add 5 μl Recombinant plasmid (recombinant clone T vector), ice bath for 30min, heat shock in 42°C water bath for 90s, place on ice for 2min, add 500μl LB liquid medium and mix well, take 200μl and spread on LB plate containing Amp (60μg / ml) above, cultured at 37°C for 12 hours, picked a single colony and cultured it in LB liquid medium to ensure the integrity of the gene fragment; extra...

Embodiment 3

[0161] Evaluation of the safety and immune protection effect of enterotoxin STa-LTB-STb fusion protein on experimental animals

[0162] (1) Ultrafiltration concentration of recombinant enterotoxin STa-LTB-STb fusion protein

[0163] Concentrate the supernatant from the induced culture of the recombinant strain of Pichia pastoris using an ultrafiltration tube, and operate according to the instructions of the Amicon Ultra-15 ultrafiltration centrifuge tube produced by Millipore, USA. First, the culture medium is centrifuged at 2000rpm for 5min; the obtained supernatant is added to an ultrafiltration tube, centrifuged at 4000rpm for 25min, the filtrate is discarded, and the collected solution is stored for later use, which is enterotoxin STa-LTB-STb Fusion protein solution, the SDS-PAGE electrophoresis figure of described solution is as follows figure 1 shown.

[0164] (2) Preparation of recombinant enterotoxin STa-LTB-STb fusion protein vaccine

[0165] The recombinant entero...

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Abstract

The invention discloses a STa-LTB-STb fusion protein of enterotoxin of escherichia coli. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.2 and the gene sequence of the fusion protein is as shown in SEQ ID NO.1. The invention also discloses the application of the Ta-LTB-STb fusion protein of enterotoxin in preparing a protein vaccine for preventing or treating escherichia coli induced diarrhea. The STa-LTB-STb fusion protein of enterotoxin has thermal stable enterotoxin and thermosensitive enterotoxin immunogenicity simultaneously. The STa-LTB-STb fusion protein of enterotoxin is prepared into a vaccine as an antigen to immune a mammal, high level thermal stable enterotoxin and thermosensitive enterotoxin antibodies can be generated for the body, so that the protecting effect of the vaccine is improved, and harms of enterotoxigenic escherichia coli to production of animal husbandry are prevented effectively.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to an Escherichia coli enterotoxin STa-LTB-STb fusion protein, its coding gene and application. Background technique [0002] Escherichia coli, formerly known as Escherichia coli, is one of the most common intestinal pathogens, often present in the intestinal tract of warm-blooded animals. Most E. coli species are harmless, but some strains can cause severe food poisoning symptoms in hosts, including diarrheal illness. The present invention selects a kind of pathogenic Escherichia coli——Enterotoxigenic Escherichia coli (ETEC) as the main research object, the main reason is that ETEC is one of the main pathogenic bacteria causing bacterial diarrhea in people in developing countries, and it is also the cause of infantile diarrhea. Major pathogen of diarrhea in young children, travelers and young animals, causing mass morbidity and mortality in piglets and calves. Therefore, it i...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19A61K39/108A61K39/39A61P31/04A61P1/12C12R1/84
CPCA61K39/0258A61K39/39A61K2039/552A61K2039/55544A61P1/12A61P31/04C07K14/245C07K2319/55C12N15/815
Inventor 王丽丽赵红徐永平李晓宇李根
Owner DALIAN UNIV OF TECH
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