Stable polypeptide targeting SARS-CoV-2 spike protein and application of stable polypeptide

A technology of pneumonia virus and spike protein, applied in the field of bioengineering, can solve the problems of poor effect of new coronary pneumonia disease, and achieve the effect of broadening the scope of application

Active Publication Date: 2021-02-05
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In view of the above-mentioned technical problems in the prior art, the present invention provides a stable polypeptide targeting the spike protein of the new coronary pneumonia virus and its use, and the stable polypeptide targeting the spike protein of the new coronary pneumonia virus and its use To solve the technical problem that the drugs in the prior art are not effective for the treatment of new coronary pneumonia

Method used

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  • Stable polypeptide targeting SARS-CoV-2 spike protein and application of stable polypeptide
  • Stable polypeptide targeting SARS-CoV-2 spike protein and application of stable polypeptide
  • Stable polypeptide targeting SARS-CoV-2 spike protein and application of stable polypeptide

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Embodiment 1

[0036] The present invention uses previous literature reports ((Zhao, Liu et al.2016, Tian, ​​Yang et al.2017, Wang, Yu et al.2019)) to modify the polypeptide targeting the spike protein, through a section of known activity Sequence (Linker-IEEQAKTFLDKFNHEAEDLFYQS-CONH 2 ) structural transformation, it is expected to obtain an antiviral polypeptide targeting RBD with better druggability, higher stability and higher activity than the original polypeptide sequence.

[0037] In the modification process of the polypeptide, the selection of the polypeptide structure plays a key role, mainly the screening of its length. Scholars in the field have identified that in the 23 peptide, the first 7 amino acids play a decisive role and form a hydrogen bond network with ACE2. And the 8th-17th amino acid lacks the specific binding with human ACE2, and does not. In this study, we screened for the length of the linker. We synthesized three molecules with linkers of different lengths, as sho...

Embodiment 2

[0047] Preparation and separation and purification steps of the polypeptide of embodiment 2:

[0048] According to the amino acid sequence of solid-phase synthesis of polypeptides, the core steps of preparing the above stable polypeptides are as follows (taking TD-PROTAC as an example):

[0049]

[0050] The specific operation steps are:

[0051] (1) Polypeptide solid-phase synthesis: Weigh Rink amide MBHA resin into a peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 30 minutes. Add 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 30 minutes, and remove the Fmoc protecting group. After washing the resin alternately with DMF and DCM, the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea Fluorophosphate ester (HCTU) (5eq, 0.38M, DMF) solution and N,N-diisopropylethylamine (DIPEA) (10eq) were mixed well, then added to the resin and blown with nitrogen for 1h. Take out the r...

Embodiment 3

[0056] Example 3 Expression of Spike Protein RBD Region

[0057] After RBD is expressed, it is deposited in the precipitate after broken cells. In this project, the RBD solution was obtained through the denaturation and renaturation experiments of RBD. Wherein, the buffer solution of denaturation is the guanidine hydrochloride of 6M of pH=7.2, and the buffer solution of refolding is the guanidine hydrochloride of 3M~0M of pH=7.2 ( Image 6 , Figure 7 ). However, the obtained protein has no activity, and the FP experiment shows that there is no obvious combination with the polypeptide.

[0058] In order to solve the problem that the prokaryotic expressed protein is inactive, we constructed an insect expression system for the RBD region of the spike protein and purified the corresponding protein. Figure 8 The gel image of the purified RBD protein is shown, and it can be seen that we obtained RBD protein with high purity through Ni column purification. We used the RBD prote...

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Abstract

The invention provides a stable polypeptide targeting SARS-CoV-2 spike protein. A template amino acid sequence structure of the stable polypeptide is X-(Linker)-CONH-IEEQAKTFLDKFNHEAEDLFYQS-CONH2. Theinvention also provides application of the polypeptide in preparation of a medicine for treating SARS-CoV-2. The invention also provides application of the polypeptide in preparation of a medicine for preventing the SARS-CoV-2 from entering human cells. The polypeptide molecule is combined with the spike protein to block virus from entering the human body by being combined with human ACE2. Experiments of fluorescence polarization detection and the like prove that the polypeptide disclosed by the invention can be well combined with an RBD of the spike protein and has an effect of blocking a virus infection process.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, specifically a stable polypeptide targeting the spike protein of the novel coronavirus pneumonia virus and its application. Background technique [0002] Since the end of 2019, the new type of coronavirus has caused a global pandemic in a short period of time due to its extremely high infectivity, and its number of infections and deaths far exceeds that of SARS and Middle East Respiratory Syndrome (MERS). Epidemics caused by species of coronavirus. On March 11, WHO declared the new crown a "global pandemic" (Pandemic). Remdesivir is approved in Japan to treat patients infected with the novel coronavirus. Outside of Japan, remdesivir is a research drug that has not yet been approved. The U.S. Food and Drug Administration granted Remdesivir an emergency use authorization (EUA) for the treatment of severe hospitalized patients with new coronavirus pneumonia; the authoriz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00A61K38/16A61P31/14
CPCC07K14/00A61P31/14A61K38/00Y02A50/30
Inventor 李子刚尹丰周子原乔坤施琴朗严尔福
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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