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Binding protein for labeling beta-D glucan and purifying and synthesizing method of binding protein

A technology of combining proteins and synthetic methods, applied in the field of biomedical diagnosis, can solve problems such as limited application scope, and achieve the effects of convenient clinical use, improved detection efficiency, and improved fungal detection efficiency

Inactive Publication Date: 2018-09-14
江苏诺鬲生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, histopathological staining can only identify some fungal species, and the scope of application is limited

Method used

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  • Binding protein for labeling beta-D glucan and purifying and synthesizing method of binding protein
  • Binding protein for labeling beta-D glucan and purifying and synthesizing method of binding protein
  • Binding protein for labeling beta-D glucan and purifying and synthesizing method of binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Protein prokaryotic expression experiment

[0029] Expression host: choose E.coliRosetta as the host expression strain.

[0030] Expression conditions: 37°C, shake culture at 220rpm, when OD600=0.4-0.6, add IPTG with a final concentration of 0.5mM, induce expression at 18°C ​​for 10 hours; see Figure 4 , Figure 5 .

Embodiment 2

[0031] Example 2 Culture method and elution and purification after protein induction and expression, inoculate the specimen into the culture medium, take high-expression strains, mix with Sabao agar cooled to about 45°C, and pour into the inoculation plate. They were placed in 35°C and 25°C incubators for simultaneous cultivation. Cultivate at 35°C for 48h, and at 25°C for 5 days, observe the results daily. Suspicious colonies of fungi and yeast were found, and they were transferred to the slant of Sabaobab. After obtaining pure culture, protein expression was induced and eluted and purified.

[0032] The results show that the protein can be hung on the column during purification, and the eluted protein is less. When doing it in batches, it is necessary to ①increase the digestion time, ②increase the DTT concentration, and ③elution 2-3 times. Currently about 300ug; see Figure 6 .

Embodiment 3

[0033] Example 3 Antigen labeling experiment of clinical fungal specimens

[0034] Select 3 leucorrhea specimens with similar size, shape and weight and place them on glass slides respectively, respectively label specimens 1, 2, and 3 with antibody protein and fluorescent whitening agent, place them at room temperature for 3 minutes, add a cover glass, and then place them on the glass slide. Observed under a fluorescence microscope. Activated with 340nm–400nm UV light, the fungus will appear blue or blue-white. The results are shown in Table 1. The results showed that the three specimens all had good marking effects, and the photographs were taken in order Figure 1-Figure 3 .

[0035]

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Abstract

The invention discloses a binding protein for labeling beta-D glucan and a purifying and synthesizing method of the binding protein. The purifying and synthesizing method of the binding protein comprises the steps of transformation of a vector system and optimized induction to an expression strain, screening and culturing on the high-expression strain after a small amount of tests, and a large amount of protein purification, synthesis and elution. The constructed expression vector is transformed into the expression strain, bacterial colonies are screened and PCR sequencing verification is carried out to obtain a positive strain, after a small amount of preliminary experiments are carried out, SDS-PAGE is carried out meanwhile to confirm expression of a target protein and screen out the high-expression strain, then a large number of high-expression strains are cultured, and a large amount of protein purification is further carried out. The binding protein and beta-D glucan as a fungal cell wall can be subjected to immunoaffinity effectively, and fungal surface beta-D glucan antigen is labeled accurately.

Description

technical field [0001] The invention belongs to the technical field of biomedical diagnosis, and specifically relates to a binding protein capable of marking fungal cell wall component β-D glucan and a purification and synthesis method thereof. Background technique [0002] Fungi are widely distributed in nature, most of which are harmless or even beneficial to humans, but more than 400 of them can cause human diseases, which are what we call pathogenic fungi. Pathogenic fungi can be divided into dermatophytes, yeasts and molds. Diseases caused by fungi are called mycoses. Clinically, mycosis can be divided into superficial mycosis and deep mycosis or systemic mycosis. Superficial mycoses are caused by fungi that only invade superficial tissues such as human skin, hair, and nails. More than 90% of mycoses in my country are superficial mycoses. Pathogenic fungi that cause systemic mycoses include Histoplasma, Blastomyces, Coccidioides, and Paracoccidioides, which are bipha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K16/14G01N33/569
CPCC12N15/70C07K16/14G01N33/569G01N2469/10
Inventor 咸涛
Owner 江苏诺鬲生物科技有限公司
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