CN115300608A

CN115300608A - Method for blocking new coronavirus infection by using mannose-binding lectin

Info

Publication number: CN115300608A
Application number: CN202110490146.9A
Authority: CN
Inventors: 鲍锦库; 吴传芳; 曹东炜; 郑茹潇; 刘美林; 王意达; 陈宣铭
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-05-06
Filing date: 2021-05-06
Publication date: 2022-11-08
Anticipated expiration: 2041-05-06
Also published as: CN115300608B

Abstract

The invention discloses an application of human Mannose-Binding Lectin (Mannose-Binding Lectin) in resisting novel coronavirus, which is characterized in that a protein sequence is shown as a sequence table SEQ NO. 1. Antiviral experiments show that recombinant expressed MBL can effectively block the infection of VERO cells by the novel coronavirus. Therefore, the human mannose binding protein can be used as an active ingredient for preparing a medicament for resisting the novel coronavirus.

Description

Method for blocking new coronavirus infection by using mannose-binding lectin

Technical Field

The invention belongs to the field of biological medicine, and is especially human plasma protein-mannose binding protein blocking new type coronavirus (SARS-CoV-2) invasion and infected cell blocking method.

Background

The process of infecting cells by the novel coronavirus is mainly mediated by the combination of S protein (Spike) on the surface of the coronavirus and angiotensin converting enzyme 2 (ACE 2) on epithelial cell membranes of organ tissues such as nasal cavities, lungs, small intestines and the like of a host. The S protein usually forms "spikes" on the virus surface in the form of homotrimers, which bind ACE2, causing the virus to fuse with the host cell membrane and enter the cell. Therefore, blocking the binding of the S protein to the ACE2 protein can be a target for treating new coronary pneumonia. The novel coronavirus S protein is a highly glycosylated protein, a monomer comprises at least 22N-linked sugar chains, the S protein of a trimer comprises at least 66 glycosylation sites, a large number of oligomannose, high mannose and mannose-containing complex sugar chains are attached to the surface, and the sugar chains form a barrier on the surface of the virus, can shield an epitope and prevent the combination of an antibody and the virus, thereby causing immune escape and drug off-target effect. Meanwhile, researches show that some sugar chains on the surface of the novel coronavirus can stabilize the open conformation of the S protein, thereby promoting the binding of the S protein and ACE 2. Therefore, novel coronavirus surface carbohydrate chains are key to their immune escape and display high affinity for ACE 2. Therefore, blocking targeting the S protein sugar chain on the surface of the novel coronavirus is an effective virus inhibition means.

Mannose-binding proteins are members of the C-type lectin superfamily, and endogenous collagen-like glycoproteins secreted by hepatocytes are important pattern recognition receptors. MBL is an acute phase reaction protein, is mainly secreted in serum, and plays an important role in nonspecific immune response. The mature MBL peptide chain is sequentially provided with an N-terminal region rich in cysteine (Cys), a collagen-like region CLR, a neck region of an alpha helical structure and a carbohydrate recognition region CRD at the C terminal from the N terminal to the C terminal. In a natural immune system, MBL has an important biological regulation function, CRD can be selectively combined with ligands which are rich in mannan oligosaccharides on the surfaces of pathogenic microorganisms, necrotic damaged cells, apoptotic cells, tumor cells and the like, CLR is combined with mannan-related serine protease MASPs to form an MBL-MASPs compound, and a complement lectin pathway is activated, so that the immune regulation function is exerted; can also bind to specific gelator receptors (C1 qR) on the surface of phagocytes to initiate neutrophil-mediated phagocytosis; can also protect human body by stimulating cell to secrete cytokine. Research shows that MBL has obvious inhibition effect on SARS-CoV-1, HIV, influenza A virus, etc.; however, the inhibition of the novel coronavirus SARS-CoV-2 by MBL has not been reported. Therefore, the invention determines the S protein binding activity and SRAS-CoV-2 antiviral activity of MBL, and finds that the MBL can specifically and efficiently bind with S protein sugar chains; antiviral activity experiments show that the sealwort lectin protein can effectively block the infection of novel coronavirus on VERO cells, and the antiviral ability of the lectin protein is greatly reduced after mannose is added to seal MBL protein mannose binding sites. Therefore, the MBL protein can be used as an active ingredient to prepare a medicament for resisting the novel coronavirus. The invention provides possibility for a novel blocking method for blocking the invasion infection of the novel coronavirus SRAS-CoV-2 by using MBL protein so as to achieve the effect of blocking and preventing the virus infection.

Disclosure of Invention

The invention aims to provide a method for blocking novel coronavirus invasion infection by using human serum mannose-binding lectin protein.

It is still another object of the present invention to provide the above use of human serum mannose binding lectin protein for blocking invasion of novel coronavirus.

The purpose of the invention is realized by the following modes:

(1) Expression of Mannose Binding Protein C (Mannose Binding Protein-C) was purified.

(2) Performing a virus TCID50 (tissue median infectious amount) assay, and examining the antiviral activity of mannose binding protein of the protein sample to be tested in step (1).

The invention has the beneficial effects that: human endogenous protein is used for virus blocking, and compared with small molecular drugs and heterologous protein, the vaccine has the advantages of lower immunogenicity, smaller side effect, low development cost and easy acquisition; can be specifically targeted and combined with oligomannose type, high mannose type and mannose containing complex type sugar chains with a plurality of coronavirus surfaces. Different from traditional targeted drugs such as vaccines and antibodies, the virus blocking targeting of the surface sugar chain of the novel coronavirus based on the mannose binding protein can solve the problem of immune escape of the virus by utilizing the surface sugar to cover an epitope and amino acid sequence mutation to a certain extent. Therefore, the mannose binding protein can be used for blocking viruses by binding mannan sugar chains on the surface of the novel coronavirus, and blocking the recognition and binding of the viruses and host cells, so that the novel coronavirus is prevented from infecting the host cells, and the aim of efficiently resisting viruses with low toxic and side effects is fulfilled.

Drawings

FIG. 1: purified MBL is detected by Coomassie brilliant blue staining;

FIG. 2 is a schematic diagram: MBL inhibition curve for new coronavirus infection.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1: expression and purification of recombinant mannose-binding protein

Materials: the pCMV-MBL2 plasmid (Yi Qiao Shen); HEK293T cells; manna-agarose resin (Sigma); ITS-A (source culture); l-ascorbic acid (BBI).

(1) Transfection of HEK293T cells with pCMV-MBL2 plasmid 6h later the medium was replaced by RPM-1640, adding 1% ITS and 50. Mu.M ascorbic acid at final concentration, 5% CO at 37 ℃% ₂ After 96h of culture, the culture solution is collected, and 4000g of culture solution is centrifuged to remove cell sediment, which is called as a sample crude product.

(2) With TBS (containing 10mM CaCl) ₂ ) The Manna-agarose resin was equilibrated and washed thoroughly and mixed with the crude sample in a vertical homogenizer at 4 ℃ for 16h.

(3) The resin and sample mixture are loaded onto a chromatographic column for separation with TBS-Ca ²⁺ After washing well (about 15-20 column volumes), elution was performed with 20mM EDTA.

(4) The eluate was concentrated by centrifugation using 4000g of a 30kD ultrafiltration tube.

(5) The TBS was dialyzed against the concentrate and examined by SDS-PAGE gel electrophoresis (FIG. 1, examination of purified MBL by Coomassie blue staining).

Example 2: experiment for blocking novel coronavirus by recombinant mannose binding protein

1. Cell preparation: 1 day before experiment, good Vero cells were taken, digested with pancreatin, counted, diluted with complete medium and prepared to 1.5X 10 ⁵ Cell suspension at/mL concentration, 100. Mu.L of cell suspension was added to each well of a 96-well cell culture plate, and placed in CO ₂ The cells were incubated overnight in an incubator and observed for good cell status before infection, and then washed 2-3 times with TBS, and 100. Mu.L of maintenance medium was added to each well.

2. The MBL pure product is treated with TBS (containing 2mM CaCl) ₂ ) Diluted to 20. Mu.g/mL as initial concentration; and MBL-free wells were set as control groups.

3. Sample dilution: diluting samples according to a two-fold ratio, diluting the samples in a 96-hole U-shaped plate, wherein each sample needs at least two times, and each to-be-detected hole is ensured to have a to-be-detected sample with the volume of 40 mu L after dilution.

4. Virus incubation with MBL samples: taking virus with known TCID50 titer, diluting the virus with a maintenance solution to the concentration of 100 TCID50/35 muL (which can be adjusted according to experiment needs, and part of experiments are 200 TCID50/35 muL), then adding virus diluent with the volume of 35 muL into each MBL sample hole, placing the MBL sample hole in a safety cabinet for incubation for 2 h at room temperature, preparing a Vero cell plate during the incubation, washing the Vero cell plate twice with PBS, measuring incubation mixed liquor of the virus and the sample according to the volume of 35 muL/hole after the incubation is finished, adding the incubation mixed liquor into the cells according to corresponding holes, and placing the MBL sample hole in a CO2 incubator for incubation for 2 h at 37 ℃.

5. Adding 120 mu L of virus maintenance solution into each cell culture hole, placing the cell culture hole in a cell culture box at 37 ℃ for 3-5 days, observing the virus inhibition, and calculating the IC50 to obtain the IC50=25 mu g/mL (FIG. 2, MBL on the new coronavirus infection inhibition curve).

Sequence listing

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Pro Ala Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly

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Gly Glu Ile Met Thr Phe Glu Lys Asn Arg Leu Thr Tyr Thr Asn Trp

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Leu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His

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Leu Ala Val Cys Glu Phe Pro Ile

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Claims

1. An application of protein in resisting novel coronavirus infection, which is characterized in that the protein is human serum mannose-binding lectin, and the sequence of the protein is shown as a sequence table SEQ NO. 1.

2. Use according to claim 1, characterized in that the inhibition of the novel coronavirus by the protein MBL is achieved by blocking its invasion of the cells.

3. The use according to claim 1, wherein the MBL protein is derived from recombinant expression, allowing for limited production of MBL protein from human serum.