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Polypeptide specially bound with mycobacterium tuberculosis, and coding gene and application of polypeptide

A Mycobacterium tuberculosis, species-specific technology, applied in the field of biomedicine, can solve problems such as unsatisfactory diagnosis and analysis, and achieve the effects of low cost, simple preparation process and high stability

Active Publication Date: 2020-08-11
NINGXIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although MA is closely related to the pathogenesis of Mycobacterium tuberculosis and can be an ideal diagnostic antigen, MA detection methods usually require highly sophisticated instruments and well-trained personnel, and are not suitable for diagnostic analysis in resource-poor settings. not ideal
[0005] In general, the existing laboratory diagnostic methods still have many limitations, and it is necessary to find a fast, accurate, economical and convenient diagnostic method

Method used

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  • Polypeptide specially bound with mycobacterium tuberculosis, and coding gene and application of polypeptide
  • Polypeptide specially bound with mycobacterium tuberculosis, and coding gene and application of polypeptide
  • Polypeptide specially bound with mycobacterium tuberculosis, and coding gene and application of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Screening of polypeptides that bind to MA

[0055] 1. Amplification and purification of phage library

[0056] inoculation E. coli ER2738 single colonies were incubated in 5-10mL LB liquid medium, 37°C, 200rpm shaker to mid-logarithmic phase (OD 600 ≈0.5); add 10 μL of phage, shake at 37°C and 200rpm for 4-5h, centrifuge at 10,000g for 10min, and take the supernatant; centrifuge again at 10,000g for 10min, take 80% of the supernatant, add 1 / 6 volume of PEG8000 / NaCl, 4°C Let stand overnight. Take it out the next day, and the white precipitate is the phage. Centrifuge at 10000g for 15 minutes, discard the supernatant, and gently suck out the residual solution after brief centrifugation; add 1mL TBS solution to dissolve the white precipitate.

[0057] 2. Phage Titer Determination

[0058] inoculation E. coli ER2738 single colonies were incubated in 5-10mL LB liquid medium at 37°C and 250rpm on a shaker until mid-log phase (OD 600 ≈0.5); Microwave heati...

Embodiment 2

[0065] Example 2 Identification of MA-specific phage

[0066] Randomly pick 20 well-separated single colonies on the titer determination plate after the third round of screening in Example 1, and after culturing and purifying separately, use phage ELISA to detect the binding activity of each strain of phage to MA. The specific steps are as follows:

[0067] Coat the ELISA plates with MA and Blocking respectively, and each group was paralleled three times, and blocked overnight at 4°C, washed 6 times with TBST, added 100 μL of purified phage, incubated at 37°C for 1 hour with shaking, washed 10 times with TBST to remove unbound For phage, add 100 μL of HRP-labeled mouse anti-M13 monoclonal antibody, incubate with shaking at 37°C for 1 hour, wash 10 times with TBST, add TMB substrate and react in the dark at room temperature for 5-10 minutes, stop the reaction with 2mol / L sulfuric acid, and measure the OD value at a wavelength of 450nm , with P / N ≥ 2.1 as positive.

[0068] Te...

Embodiment 3

[0073] Example 3 Determination of binding activity of polypeptide sequences

[0074] The plates were coated with different concentrations of MA and Blocking and blocked overnight at 4°C, washed 6 times with TBST, and 2×10 phages with the Thanos1 sequence were added. 11 (with the same titer of phage without Thanos1 sequence as the control), wash 10 times with TBST to remove unbound phage, add 100 μL of HRP-labeled mouse anti-M13 monoclonal antibody, incubate with shaking at 37°C for 1 hour, wash 10 times with TBST; TMB was reacted at room temperature in the dark for 5-10 minutes, 2mol / L sulfuric acid was used to terminate the reaction, and the OD value was measured at a wavelength of 450nm. The result is as figure 2 As shown, phages with the Thanos1 sequence exhibited specific binding to MA.

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Abstract

The invention discloses polypeptide specially bound with mycobacterium tuberculosis, and a coding gene and application of the polypeptide, and belongs to the field of biomedical science. The polypeptide comprises an amino acid sequence as shown in SEQID No.2, and the coding gene comprises a nucleotide sequence as shown in SEQID No.1. The invention further discloses an application of the polypeptide to preparation of a reagent kit for detecting disinfection of the mycobacterium tuberculosis or medicines for targeting the mycobacterium tuberculosis. The polypeptide can be used for diagnosing tuberculosis, has high diagnosis sensitivity and specificity and has great application prospects, and a new method is provided for targeted diagnosis of the tuberculosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a polypeptide specifically binding to Mycobacterium tuberculosis, its coding gene and application. Background technique [0002] Mycobacterium tuberculosis( Mycobacterium tuberculosis , M.tb ) infection caused by tuberculosis (Tuberculosis, TB) is a serious threat to public health security worldwide infectious disease. The unique cell envelope lipids produced by Mycobacterium tuberculosis play a crucial role in the pathogenicity of these bacteria. The cell membrane of mycobacteria is composed of lipids and proteins surrounded by a complex cell wall composed of carbohydrates and lipids. The cell wall of Mycobacterium tuberculosis and other mycobacteria has an unusual structure comprising a multilayered and extremely hydrophobic envelope, which is essential for the survival of the organism in macrophages. [0003] Early diagnosis is the first step in the treatment and ter...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11C12N7/00G01N33/68G01N33/569A61K38/08A61P31/06
CPCA61K38/00A61P31/06C07K7/06C12N7/00G01N33/5695G01N33/68G01N2410/00
Inventor 薛頔林雪魏萌萌王玉炯
Owner NINGXIA UNIVERSITY
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