Application of spd_0310 protein as a target in the preparation of drugs for preventing and treating Streptococcus pneumoniae infection

A Streptococcus pneumoniae and drug technology, applied in the field of protein science, can solve the problems of low detection sensitivity, low expression level, and undetectability, and achieve high conservation, strong toxicity, and good broad-spectrum effects

Active Publication Date: 2022-01-18
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of detection, the current understanding of the virulence proteins of this bacteria is still on the adhesion factors such as PspA, Ply, PcpA, CbpA and CbpD, and the expression levels of these five proteins in the bacteria are generally low. When the amount of viable bacteria is small, the detection sensitivity of the above adhesion factors is low or even impossible to detect. Since there are no antibody drugs on the market for the above five proteins, the detection process is time-consuming and laborious, and it is easy to cause false positives. Therefore, it is necessary to develop a New virulence protein markers
In terms of prevention, it mainly relies on the vaccination of PPV23 and PCV7. The disadvantage of this measure is that it can only be effective against specific serotypes of Streptococcus pneumoniae, and it cannot really achieve the purpose of prevention. timely control

Method used

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  • Application of spd_0310 protein as a target in the preparation of drugs for preventing and treating Streptococcus pneumoniae infection
  • Application of spd_0310 protein as a target in the preparation of drugs for preventing and treating Streptococcus pneumoniae infection
  • Application of spd_0310 protein as a target in the preparation of drugs for preventing and treating Streptococcus pneumoniae infection

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Experimental program
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Effect test

Embodiment 1

[0070] The specific combination of embodiment 1SPD_0310 protein and hemin

[0071] We successively explored the SPD_0310 protein and Fe 3+ , heme (hemin, purchased from Sigma-Aldrich), fch (iron pigment, purchased from Sigma-Aldrich) and Mn 2+ The binding status of the protein, that is, the protein was incubated with the above five ligand small molecules for 6 hours, and then the chelated PBS buffer was used for overnight dialysis treatment for 3 times. Finally, we found that other metals failed to bind through ICP-MS detection. For 0310 protein, only 0310-hemin has an iron content detection value of 7.524 mg / L, which is 134 μM, while the protein concentration is 30 μM. It can be seen that the binding ratio of SPD_0310 protein to hemin is about 1:4, which indicates that hemin may be the binding substrate of the protein.

[0072] In order to further verify this inference, we first incubated 4 μg of 0310 protein with hemin-Agarose and apo-Agarose magnetic beads (purchased from...

Embodiment 2

[0074] Example 2 Surface plasmon resonance (SPR) detects the binding of SPD_0310 protein to hemin

[0075] Utilize the Open-SPR instrument (premedi, nicoya, Canada) to detect the combination of protein and ligand hemin, with 1 × PBS buffer as mobile phase buffer, with 0.2M N-hydroxysuccinimide (EDC) and The 50mM N-ethyl-N'-(diethylaminopropyl)-carbodiimide (NHS) 1:1 (volume ratio) mixed solution will be purchased for the nano-gold-COOH sensor chip (premedi, nicoya, Canada) surface amino group activation, diluted with 10nM sodium acetate buffer solution of pH 4.5 to a final concentration of about 50 μg / mL, injected at a flow rate of 30 μL / min for 500 s, and then injected the diluted SPD_0310 protein solution with 1M ethanolamine pH 8.5 The blocking solution was injected for 7 minutes to block the activated chip surface, that is, the SPD_0310 protein was tightly combined with the amino group of the chip through the mobile phase. Dilute hemin with sample diluent to different con...

Embodiment 3

[0077] CD spectrum detection of embodiment 3SPD_0310 protein binding to Hemin

[0078] The detection results of the above SPR experiments suggest that the main biological function of the SPD_0310 protein is the binding and transport of hemin. So, will the 0310 protein cause a change in protein conformation after binding the ligand? To this end, we used circular dichroism chromatography to detect the secondary structure (secondary-structure) of Apo-0310 and 0310-hemin respectively [5] .

[0079] Such as Figure 5 As shown, the CD peak patterns of Apo-0310 protein and 0310-hemin are almost the same. After detecting the content of various indicators of the secondary structure by CDPro software, it is found that there is no obvious difference between the two, which shows that the binding of the protein to hemin will not cause drastic changes in its secondary structure.

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Abstract

The invention discloses the application of SPD_0310 protein as a target in the preparation of medicines for preventing and treating Streptococcus pneumoniae infection. In the present invention, it was found that the expression level of SPD_0310 in the triple mutant strain in which the three main iron transport genes of Streptococcus pneumoniae were knocked out at the same time was increased and could grow normally, indicating that the SPD_0310 protein may be involved in the interaction of various iron transport proteins. The present invention also found that the spd_0310 gene knockout strain has reduced infectivity to mice. Based on this, SPD_0310 can be used to prepare antibodies to the protein, so as to facilitate the development of antibody drugs. In addition, the present invention also found that the active center of the protein can bind heme, and proved that porphyrins can be used as substrates to compete with heme, indicating that porphyrins can be used as inhibitors of SPD_0310 to inhibit Streptococcus pneumoniae toxicity, which has broad application prospects in the field of antibacterial drugs.

Description

technical field [0001] The invention belongs to the field of protein science and technology, and particularly relates to the application of SPD_0310 protein as a target in the preparation of drugs for preventing and treating Streptococcus pneumoniae infection. Background technique [0002] Streptococcus pneumoniae is a Gram-positive pathogenic bacterium, and it is also the main pathogenic microorganism that causes human infection and even death worldwide, especially for the elderly and children with weak resistance. According to the World Health Organization (WHO) According to incomplete statistics, the bacteria cause about 1.6 million deaths every year. After this bacterium infects the human body, it mainly causes serious diseases such as pneumonia, otitis media (AOM), sepsis, acute renal failure and meningitis. At present, there is no specific drug in the world that can completely inhibit the toxicity of this bacterium. Therefore, people's strategies for this bacterium ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/00A61P31/04G01N33/569A61K31/409
CPCA61K45/00A61P31/04A61K31/409G01N33/56944
Inventor 孙雪松曹锟
Owner JINAN UNIVERSITY
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