Mycobacterium tuberculosis recombinant fusion protein EECC as well as preparation method and application thereof

A technology for Mycobacterium tuberculosis and fusion protein, which is applied in fusion polypeptide, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of low specificity, increased diagnostic cost, and high dosage of antigen reagents, and achieves high specificity and reduced The lower limit of detection and the effect of good promotion and application value

Active Publication Date: 2019-11-08
扩增生物科技(北京)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PPD antigen shares common antigens with almost all mycobacteria. Therefore, the PPD skin test still has the problem of low specificity. For BCG immunized countries, when PPD is used as a tuberculosis epidemiological investigation, it is impossible to distinguish tuberculosis infection from BCG vaccination, or exposed mycobacterial infection caused by
The third-generation skin test reagent is a recombinant protein, and the expression of BCG is lower than that of tuberculosis protein 38KD for the diagnosis of tuberculosis, which can improve the specificity of diagnosis (He XY, et al., Scand J InfectDis, 2008; for tuberculosis diagnosis Mycobacterium tuberculosis protein, patent number: ZL200410044568.X), but there is a certain overlap with BCG vaccination
However, in the current clinical diagnosis, the amount of antigen reagents used in the skin test to diagnose tuberculosis is relatively high, resulting in increased diagnostic costs

Method used

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  • Mycobacterium tuberculosis recombinant fusion protein EECC as well as preparation method and application thereof
  • Mycobacterium tuberculosis recombinant fusion protein EECC as well as preparation method and application thereof
  • Mycobacterium tuberculosis recombinant fusion protein EECC as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of Mycobacterium tuberculosis recombinant fusion protein EECC

[0054] According to the published genome sequence of Mycobacterium tuberculosis H37Rv (http: / / genolist.pasteur.fr / Tubercu List / ), the amino acid of ESAT6 protein (referred to as E protein) (as shown in SEQ ID NO.5) and the base of the coding gene were obtained base sequence (as shown in SEQ ID NO.6); and the amino acid sequence of CFP10 protein (referred to as C protein) (as shown in SEQ ID NO.7) and the base sequence of the coding gene (as shown in SEQ ID NO.8). The ESAT6 protein and the CFP10 protein are connected in series in the manner of ESAT6-ESAT6-CFP10-CFP10 to obtain the amino acid sequence of the EECC protein (as shown in SEQ ID NO.1) and the base sequence of the coding gene (as shown in SEQ ID NO.3) . The sequence shown in SEQ ID NO.3 was entrusted to Shanghai Bioengineering Co., Ltd. for codon optimization, and an NcoI restriction site (CCATGG) was added to the 5' end of t...

Embodiment 2

[0056] Example 2 EECC protein extraction, purification and identification

[0057] Cultivate pET28a-EECC / BL21(DE3) engineering bacteria and induce expression of EECC protein. The extraction and purification methods of EECC protein are as follows:

[0058] 1. Bacteria fragmentation

[0059] Pressurize the high-pressure homogenizer gradually to 700±50bar. When the pressure reaches the requirement, put the liquid outlet pipe into the empty beaker, which is the first crushing liquid. The crushing operation was repeated three times, and the cell crushing liquid was collected.

[0060] 2. Dilution and filtration

[0061] Dilute the centrifuged supernatant by 2-3 times the volume with the bacterial cell disruption buffer, and control the pH of the feed solution between 7.1-7.5. Filtration was performed using a 0.45 μm Bursafil filter. Collect the filtrate.

[0062] 3. Anion exchange chromatography (Capto Q)

[0063] The eluted sample was loaded at a flow rate of 300mL / min, and ...

Embodiment 3

[0074] The skin test of embodiment 3 Mycobacterium tuberculosis sensitized guinea pigs

[0075] Healthy SPF grade white guinea pigs without any experiments, weighing 300g-500g. Take out one tube of frozen Mycobacterium tuberculosis, dissolve it naturally at room temperature, and dilute it 10 times with normal saline; inject 0.5ml of the diluted bacteria solution subcutaneously into the groin of each guinea pig’s hind leg, and test the skin of the guinea pig 5-6 weeks after sensitization. The guinea pigs were depilated on both sides of the spine, and 0.2ml of EECC samples of each dilution concentration and TB-PPD and EC standards were injected intradermally. The double-blind method measures the longitudinal diameter and transverse diameter (mm) of redness and / or induration at the injection site respectively at 24 and 48 hours, and the mean value of the longitudinal diameter and transverse diameter is used as the skin test reaction diameter of the injection sample at this point....

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Abstract

The invention relates to the biotechnology field, in particular to mycobacterium tuberculosis recombinant fusion protein EECC as well as a preparation method and application thereof. The mycobacteriumtuberculosis recombinant fusion protein EECC is EAST6-EAST6-CFP10-CFP10, with an amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2. The recombinant fusion protein EECC has excellent antigenicity, has higher sensitivity while guaranteeing higher specificity during diagnosis of tuberculosis, can effectively reduce the use dosage, reduce the detection cost and effectively distinguish and diagnose viable bacterium infection of tubercle bacillus, dead bacterium sensitization and BCG inoculation, can be applied to diagnosis of the tuberculosis, preparation of tuberculosis vaccines and detection of antigen specific cell factors and has better promotion and application values.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a Mycobacterium tuberculosis recombinant fusion protein EECC prepared by genetic engineering technology, a nucleic acid encoding the protein, a vector containing the nucleic acid and a host cell, as well as a preparation method and application of the fusion protein . Background technique [0002] Diagnosis methods for tuberculosis targeting pathogenic bacteria include acid-fast staining microscopy, strain culture, molecular biology diagnostic methods based on PCR amplification, etc.; however, diagnostic methods targeting pathogenic bacteria are not suitable for the diagnosis of bacillus-negative tuberculosis patients The number of patients is slowly decreasing, but the number of patients with bacterium-negative tuberculosis is increasing (it has reached 80%). These patients urgently need to establish immunological diagnostic methods to distinguish tuberculosis infection from ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N1/21C12N15/70G01N33/68G01N33/569A61K39/04A61P31/06
CPCA61K39/04A61P31/06C07K14/35C07K2319/00C12N15/70G01N33/5695G01N33/6893G01N2333/35
Inventor 张鹏飞杨晰朦王化楠任永峰崔颖杰李福胜林兆新胡瞬张青乐王国治
Owner 扩增生物科技(北京)有限公司
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