Application of lentil agglutinin in the preparation of drugs for preventing and treating infectious diseases caused by coronavirus

Abstract

The invention discloses the application of lentil agglutinin in the preparation of medicines for preventing and treating infectious diseases caused by coronaviruses. The invention finds for the first time that lentil agglutinin can simultaneously treat SARS‑CoV‑2 and SARS‑CoV‑2 epidemics The mutant strain shows better antiviral activity, has significant inhibitory effect, and does not show cytotoxicity, and can be used as a candidate drug for inhibiting SARS-CoV-2 and SARS-CoV-2 epidemic mutant strains, and has broad application prospects.

Description

technical field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of lentil lectin in the preparation of medicines for preventing and treating infectious diseases caused by coronaviruses. Background technique

[0002] Coronaviruses (CoVs) are widespread in nature, and their natural hosts include humans and other mammals such as cattle, pigs, dogs, cats, mice, and bats. Coronavirus is an enveloped, non-segmented, single-stranded positive-sense RNA virus belonging to the Ortho-coronavirinae subfamily of the Coronaviridae family of the order Nidovirales. There are protrusions protruding around, which are named after the shape of a corolla. The subfamily of coronaviruses is further subdivided into four categories, namely alpha, beta, gamma and delta coronaviruses (Fehr A R, Perlman S.Coronaviruses: an overview of their replication and pathogenesis[J].Coronaviruses,2015:1-23.), at present, 7 kinds of coronaviruses th...

Examples

Embodiment 1

[0072] Example 1 Phytohemagglutinin detection of SARS-CoV-2 S protein binding activity and antiviral activity

[0073] 1. Experimental materials

[0074]Lentil lectin, wheat germ agglutinin (WGA), sessile elderberry agglutinin (SSL), peanut lectin (Peanut lectin), Huaihuai agglutinin (MAL) were purchased from Wako (Japan) Datura agglutinin (DSL), succinyl-concanavalin A (succ-Con A), galactus agglutinin (GNL), phytohemagglutinin (PHA-E), leukolectin (PHA-L ), phytohemagglutinin-M (PHA-M) and phytohemagglutinin-P (PHA-P) from red kidney bean were purchased from Sigma Aldrich; SARS-COV-2 S trimer expressed in HEK293 cells was purchased from at ACRO Biosystems (China, Beijing); 293T cells were provided by the American Type Culture Collection (ATCC); Huh7 cells were provided by the Japanese Research Biological Resource Collection (JCRB); the SARS-CoV-2 spike (original strain, MN908947; hCoV-19 / South Africa / KRISP-K007869 / 2020, B.1.351, EPI_ISL_860630; hCoV-19 / England / QEUH-F56F0F / ...

Embodiment 2

[0088] Embodiment 2 Phytohemagglutinin detects the inhibitory effect of SARS-CoV-2 S protein mutant strain and SARS-CoV-2 epidemic mutant strain infection

[0089] In this example, the inhibitory effects of various plant lectins on the infection of SARS-CoV-2 S protein mutant strains and SARS-CoV-2 epidemic mutant strains, including the British epidemic mutant strain B.1.1.7, were detected. , South African popular mutant strain B.1.351, Brazilian popular mutant strain P.1, combined mutant strain D614G+I472V, combined mutant strain D614G+A435S, A222V, K417N, S477N, E484K, N501Y, combined mutant strain A222V+D614G, combined mutant strain Mutant strains such as strain K417N+D614G, combined mutant strain S477N+D614G, combined mutant strain E484K+D614G, combined mutant strain N501Y+D614G;

[0090] Lectins are mainly aimed at the glycosyl part of glycoproteins. In this example, the inventors studied whether the change of the glycation site of SARS-CoV-2 S protein would affect its ne...

Embodiment 3

[0100] Example 3 Detection of Hemagglutination Activity and Cytotoxicity of Plant Hemagglutinin

[0101] Since many natural lectins can cause hemagglutination, in this example, the inventors studied the hemagglutination activity of different lectins, and studied the cytotoxicity of Huh7 and 293T cells.

[0102] 1. Detection of hemagglutination activity of phytohemagglutinin

[0103] Chicken blood samples were used to detect the hemagglutination activity of phytohemagglutinin, the red blood cells of the rooster were washed with PBS, and resuspended at a final concentration of 1% (v / v), and 50 μL of 2-fold serially diluted phytohemagglutinin solution was mixed with an equal volume of red blood cells The solution was mixed in a 96-well round bottom plate, and the influenza antigen (B / Maryland / 15 / 2016, NIBSC-UK-EN63QG, NIBSC code: 18 / 104, HA: 69 μg / mL) was serially diluted 2 times and used as a positive control , PBS solution was used as a negative control, the plate was incubate...

technical field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of lentil lectin in the preparation of medicines for preventing and treating infectious diseases caused by coronaviruses. Background technique

[0002] Coronaviruses (CoVs) are widespread in nature, and their natural hosts include humans and other mammals such as cattle, pigs, dogs, cats, mice, and bats. Coronavirus is an enveloped, non-segmented, single-stranded positive-sense RNA virus belonging to the Ortho-coronavirinae subfamily of the Coronaviridae family of the order Nidovirales. There are protrusions protruding around, which are named after the shape of a corolla. The subfamily of coronaviruses is further subdivided into four categories, namely alpha, beta, gamma and delta coronaviruses (Fehr A R, Perlman S.Coronaviruses: an overview of their replication and pathogenesis[J].Coronaviruses,2015:1-23.), at present, 7 kinds of coronaviruses th...

Examples

Embodiment 1

[0072] Example 1 Phytohemagglutinin detection of SARS-CoV-2 S protein binding activity and antiviral activity

[0073] 1. Experimental materials

[0074]Lentil lectin, wheat germ agglutinin (WGA), sessile elderberry agglutinin (SSL), peanut lectin (Peanut lectin), Huaihuai agglutinin (MAL) were purchased from Wako (Japan) Datura agglutinin (DSL), succinyl-concanavalin A (succ-Con A), galactus agglutinin (GNL), phytohemagglutinin (PHA-E), leukolectin (PHA-L ), phytohemagglutinin-M (PHA-M) and phytohemagglutinin-P (PHA-P) from red kidney bean were purchased from Sigma Aldrich; SARS-COV-2 S trimer expressed in HEK293 cells was purchased from at ACRO Biosystems (China, Beijing); 293T cells were provided by the American Type Culture Collection (ATCC); Huh7 cells were provided by the Japanese Research Biological Resource Collection (JCRB); the SARS-CoV-2 spike (original strain, MN908947; hCoV-19 / South Africa / KRISP-K007869 / 2020, B.1.351, EPI_ISL_860630; hCoV-19 / England / QEUH-F56F0F / ...

Embodiment 2

[0088] Embodiment 2 Phytohemagglutinin detects the inhibitory effect of SARS-CoV-2 S protein mutant strain and SARS-CoV-2 epidemic mutant strain infection

[0089] In this example, the inhibitory effects of various plant lectins on the infection of SARS-CoV-2 S protein mutant strains and SARS-CoV-2 epidemic mutant strains, including the British epidemic mutant strain B.1.1.7, were detected. , South African popular mutant strain B.1.351, Brazilian popular mutant strain P.1, combined mutant strain D614G+I472V, combined mutant strain D614G+A435S, A222V, K417N, S477N, E484K, N501Y, combined mutant strain A222V+D614G, combined mutant strain Mutant strains such as strain K417N+D614G, combined mutant strain S477N+D614G, combined mutant strain E484K+D614G, combined mutant strain N501Y+D614G;

[0090] Lectins are mainly aimed at the glycosyl part of glycoproteins. In this example, the inventors studied whether the change of the glycation site of SARS-CoV-2 S protein would affect its ne...

Embodiment 3

[0100] Example 3 Detection of Hemagglutination Activity and Cytotoxicity of Plant Hemagglutinin

[0101] Since many natural lectins can cause hemagglutination, in this example, the inventors studied the hemagglutination activity of different lectins, and studied the cytotoxicity of Huh7 and 293T cells.

[0102] 1. Detection of hemagglutination activity of phytohemagglutinin

[0103] Chicken blood samples were used to detect the hemagglutination activity of phytohemagglutinin, the red blood cells of the rooster were washed with PBS, and resuspended at a final concentration of 1% (v / v), and 50 μL of 2-fold serially diluted phytohemagglutinin solution was mixed with an equal volume of red blood cells The solution was mixed in a 96-well round bottom plate, and the influenza antigen (B / Maryland / 15 / 2016, NIBSC-UK-EN63QG, NIBSC code: 18 / 104, HA: 69 μg / mL) was serially diluted 2 times and used as a positive control , PBS solution was used as a negative control, the plate was incubate...

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