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Mycobacterium tuberculosis EEC fusion protein, and preparation method and application thereof

A Mycobacterium tuberculosis, fusion protein technology, applied in the field of preparation, Mycobacterium tuberculosis EEC fusion protein, can solve the problems of high preparation process requirements, poor repeatability of results, etc., to reduce production costs, reduce dosage, and reduce antigen extraction The effect of the purification process

Active Publication Date: 2020-01-14
成都可恩生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Patch test method has high requirements for the preparation process, and many factors affect the individual's absorption of the antigen, so the reproducibility of the results is poor

Method used

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  • Mycobacterium tuberculosis EEC fusion protein, and preparation method and application thereof
  • Mycobacterium tuberculosis EEC fusion protein, and preparation method and application thereof
  • Mycobacterium tuberculosis EEC fusion protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment one: the recombinant plasmid PET28a-EEC expressing protein EEC construction: according to Mycobacterium tuberculosis H37Rv genome (http: / / genolist.pasteur.fr / TubercuList / ), obtain E protein amino acid and coding gene base sequence (see SEQ ID NO .1 and SEQ ID NO.2) and C protein amino acid sequence and encoding gene base sequence (seeing SEQ ID NO.3 and SEQ ID NO.4), in series to obtain EEC protein amino acid sequence and encoding gene base sequence (seeing SEQ ID NO.4). ID NO.5 and SEQ ID NO.6), entrusted SEQ ID NO.6 to Shanghai Bioengineering Company to add NcoI restriction site (CCATGG) and base GC at the 5' end in sequence, and sequentially added the base GC at the 3' end A stop codon TAA and a HindIII restriction site (AAGCTT) were added. The NcoI restriction site and the HindIII restriction site are designed to clone the synthetic whole gene into the vector. After CCATGG is treated with NcoI, ATGG remains, and the genetic codes are all triplet codes. In...

Embodiment 2

[0093] Embodiment two: EEC protein (amino acid sequence as shown in SEQ ID NO.8) is expressed and identified in Escherichia coli: recombinant plasmid PET28a-EEC transforms competent host bacterium Escherichia coli BL21 (DE3), porcine resistant LB agar Plate, cultivate overnight at 37°C, pick a single colony to induce expression, and the bacteria expressing the target protein are called PET28a-EEC / BL21(DE3) engineering bacteria. The pET28a-EEC / BL21(DE3) engineered bacteria were cultured in tryptone-prepared medium to express the target protein EEC under the induction of IPTG. The pET28a-EEC / BL21(DE3) engineered bacteria in pea peptone prepared medium can express the target protein EEC in culture with or without inducer; EEC protein is expressed in soluble form in Escherichia coli.

[0094] The protein electrophoresis results of the whole bacterial body of the engineered bacteria cultivated in the peptone preparation medium of plant source and animal source are as follows: figu...

Embodiment 3

[0099] Embodiment 3: EEC protein purification strategy: the method for purifying the EEC fusion protein includes the following steps:

[0100] Step 1. Bacteria crushing: High-pressure homogenization at 600-800 Bar for 3 cycles;

[0101] Step 2. Collect the supernatant by centrifugation: 4°C, 12000-15000rpm, centrifuge for 30min-1 hour;

[0102] Step 3, supernatant sulfuric acid precipitation: the supernatant of 8-10% ammonium sulfate precipitation is precipitated with 30-35% ammonium sulfate, and the precipitate is collected;

[0103] Step 4, desalting: G-25 gel or ultrafiltration;

[0104] Step 5, anion chromatography;

[0105] Step 6: Merge the collection tubes containing the target protein, precipitate with ammonium sulfate or concentrate by ultrafiltration;

[0106] Step 7. Molecular sieve Superdex chromatography, collecting chromatographic peaks, protein electrophoresis and or HPLC detection of purity, purity > 90% collection tubes are combined;

[0107] Step 8: Combi...

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Abstract

The invention discloses a mycobacterium tuberculosis EEC fusion protein. The amino acid sequence of the fusion protein is SEQ ID NO.8 in a sequence table; polynucleotide for encoding the polypeptide is shown as SEQ ID NO.7 in the sequence table; and the fusion protein contains a carrier of the polynucleotide and host cells. The invention further relates to preparation of the fusion protein, and effects of the fusion protein in tuberculosis auxiliary diagnosis, tubercle bacillus infection screening and tuberculosis vaccine development.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mycobacterium tuberculosis EEC fusion protein, a preparation method and an application thereof. Background technique [0002] Tuberculosis is still a major public health problem, and tuberculosis control has received great attention from WHO and governments. However, nontuberculous mycobacteria (NTM) infection and the NTM disease caused by it are on the rise. In some areas with low incidence of tuberculosis, the number of NTM cases is more than that of tuberculosis. NTM is not a legally notifiable disease in my country, and its specific infection status is unknown. 80% of tuberculosis in my country is bacterial negative tuberculosis, and it is necessary to distinguish bacterial negative tuberculosis and NTM disease. At present, there is a lack of immunological detection methods for NTM infection and NTM disease. PPD-B (purified protein derivative of Mycobacterium intracellulare) is...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/569A61K39/04A61K39/116A61P31/06
CPCC07K14/35C12N15/70G01N33/5695A61K39/04A61P31/06C12N2800/22C07K2319/00G01N2333/35A61K2039/70
Inventor 张鹏飞
Owner 成都可恩生物科技有限公司
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