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Mycobacterium tuberculosis early culture filtrate protein, its preparation method and application

A technology of Mycobacterium tuberculosis and filtrate, which is applied in the field of early culture filtrate protein of Mycobacterium tuberculosis, diagnosis of tuberculosis or latent infection of Mycobacterium tuberculosis, and extraction of Mycobacterium tuberculosis H37Rv early culture filtrate protein

Inactive Publication Date: 2012-10-24
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the cultivation of Mycobacterium tuberculosis, as the number of days of cultivation increases, the protein concentration and / or composition in the filtrate protein also changes. The inventors of the present application have surprisingly found that the filtrate protein cultivated for 5 days is effective in distinguishing BCG inoculation and Mycobacterium tuberculosis infection has a more excellent effect than filtrate protein cultured for 7 days or more, this effect has not been revealed or inspired by any report

Method used

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  • Mycobacterium tuberculosis early culture filtrate protein, its preparation method and application
  • Mycobacterium tuberculosis early culture filtrate protein, its preparation method and application
  • Mycobacterium tuberculosis early culture filtrate protein, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of early culture filtrate protein

[0027] This example details H 37 Rv mycobacterium culture and preparation method of early culture filtrate protein.

[0028] 1.1H 37 Culture and inoculation of Rv mycobacterium

[0029] Will H 37 Mycobacterium Rv (ATCC 93009) was inoculated on Roche egg slant medium and cultured at 37°C for 21 days. Take the bacteria that grow well on the slope and wash the bacteria with normal saline, collect them in a centrifuge bucket, centrifuge at 4°C, 8000rpm, and centrifuge for 30min. After centrifugation, discard the culture supernatant and collect the bacteria. Wash the bacteria with liquid Sutong medium for 3 times. The washing method is to supplement the liquid Sutong medium to the volume before centrifugation, and then centrifuge to collect the bacteria. After the first washing, the protein content in the supernatant is 547μg / ml. The protein content in the supernatant after the second wash was 147μg / ml, and the protein co...

Embodiment 2

[0035] Example 2: Comparison of protein concentration of culture filtrate harvested at different culture time and its diagnostic effect

[0036] 2.1 Protein content determination

[0037] The Lowry method was used to determine the protein content (see the literature Loery OH, Rosebrough NJ, FarrAL, et al. Protein measurement with the folin phenol reagent. J Biol Chem. 1951, 193(1): 265-275).

[0038] The protein concentration in the culture filtrate harvested at different culture times obtained in Example 1 is as follows:

[0039] 0 day culture filtrate protein concentration: 18μg / ml

[0040] 3-day culture filtrate protein concentration: 33μg / ml

[0041] Protein concentration of culture filtrate for 5 days: 51μg / ml

[0042] 7-day culture filtrate protein concentration: 61μg / ml

[0043] 10 days culture filtrate protein concentration: 72μg / ml

[0044] 13-day culture filtrate protein concentration: 188μg / ml

[0045] 15-day culture filtrate protein concentration: 222μg / ml

[0046] 2.2 Analysis of...

Embodiment 3

[0055] Example 3: 5-day early culture filtrate protein as IFN- Y -Dose standardization of stimulus source for ELISPOT experiment

[0056] 3.1 Early culture filtrate protein -IFN- at different concentrations for 5 days Y -Sensitivity study of ELISPOT diagnosis

[0057] In the experiment, 24 cases of clinically confirmed tuberculosis patients were selected, and the 5-day early culture filtrate protein obtained in Example 1 with different concentrations was used as a stimulus source for in vitro ELISPOT diagnosis (the specific method is the same as that in Example 2, and the dosage is adjusted) for research Low (0.2μg / ml), medium (1μg / ml), high (5μg / ml) and other different concentrations of early culture filtrate protein in IFN- Y -Differences in sensitivity in ELISPOT diagnostic methods. The experimental results are shown in Table 2.

[0058] Table 2: Early culture filtrate protein -IFN- after 5 days of culture at different concentrations Y -Analysis of the results of ELISPOT diagnosi...

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Abstract

The invention provides a mycobacterium tuberculosis early culture filtrate protein, its preparation method and an application of the protein in reagents and kits for diagnosing tuberculosis and latent infection people of mycobacterium trberculosis. The preparation method of the protein comprises the following steps: inoculating H37Rv strain to an inclined plane medium, inoculating the cultured strain to a liquid Sauton medium, conducting shaking culturing for 5 days at 37 DEG C, and then separating.

Description

Technical field [0001] The present invention relates to early culture filtrate protein of Mycobacterium tuberculosis, its preparation method and application, in particular to Mycobacterium tuberculosis H 37 The extraction method of Rv early culture filtrate protein, and the use of the prepared early culture filtrate protein in diagnosing tuberculosis or people with latent infection of tuberculosis. Background technique [0002] Tuberculosis is a multi-organ infectious disease caused by Mycobacterium tuberculosis complex infection, mainly tuberculosis. There are up to 2 billion people infected with tuberculosis worldwide, and about 8 million to 10 million new cases occur each year. Among them, there are as many as 1.5 million new cases in my country each year. An important feature of the epidemiology of Mycobacterium tuberculosis is the latent infection of Mycobacterium tuberculosis. Latent Mycobacterium tuberculosis infection is a subclinical state with no signs of radiological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12P21/02C07K1/34G01N33/68G01N33/569C12R1/32
Inventor 王国治都伟欣陈保文徐苗沈小兵苏城杨蕾
Owner NAT INST FOR FOOD & DRUG CONTROL
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