CN115137806A - Application of Polygonatum cyrtonema lectin in blocking invasion and infection of novel coronavirus - Google Patents

Abstract

The invention discloses a sealwort agglutininPolygonatum cyrtonemaHuaLectin) protein, in the preparation of medicaments against novel coronaviruses. Antiviral experiments show that the polygonatum agglutinin protein can effectively block the infection of novel coronavirus to VERO cells, and the protein antiviral ability is greatly reduced and even lost after mannose is added to seal a polygonatum agglutinin protein sugar binding site. Therefore, the polygonatum cyrtonema agglutinin protein can be used as an active ingredient for preparing a medicine for resisting the novel coronavirus, and the specific application is that a proper amount of pharmaceutically acceptable matrix or auxiliary materials can be added, and the polygonatum cyrtonema agglutinin protein can also be combined with a proper medicine.

Description

Application of Polygonatum cyrtonema lectin in blocking invasion and infection of novel coronavirus

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a novel blocking method for blocking SRAS-CoV-2 invasive infection by using polygonatum agglutinin protein.

Background

Respiratory disease-2019 coronavirus disease (COVID-19) caused by novel coronavirus (SARS-CoV-2) is taken as the most troublesome public health problem in the world at present, and seriously jeopardizes human health and the development of the world economy and society. Currently, global scientists are actively developing vaccines aiming at novel coronavirus and novel coronary pneumonia medicines, and currently, various vaccines are put into use globally, but the protective effect of the vaccines still needs to be tested.

The novel coronaviruses mainly have four structural proteins, namely, spinous process (or spike) glycoprotein (S protein), small envelope glycoprotein (E, protein) and membrane glycoprotein (M protein), which are highly exposed on the surface of the virus, and nucleocapsid protein (N protein). The SARS-CoV-2 virus infectivity is mainly determined by S protein, it is combined with membrane receptor of host cell, and then mediates the fusion of virus and cell membrane, and the angiotensin converting enzyme 2 (ACE 2) which is widely distributed on epithelial cell membrane of organ tissues of host nasal cavity, lung and small intestine, etc. is the main receptor of S protein.

Studies have shown that the spike protein (S protein) highly exposed on the surface of the virus is the most important weapon for the virus to invade the body, is the key part for the virus to attach and infect host cells, and by forming homotrimer, the S protein forms obvious spikes on the surface of the virus, which mediate membrane fusion and virus entry into cells. Wherein the receptor Recognition Binding Domain (RBD) of the S protein is essential for its binding to ACE2 (Wrapp, D). et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science367, 1260-1263). Considering that the S protein plays an indispensable role in virus invasion and infection, the recognition and combination of the S protein in ACE2 influence the trigger of virus infected host cells, so that cutting off or interfering the interaction of the S protein and ACE2 in any way is crucial to controlling virus invasion, which is a main strategy for drug design of vaccines, neutralizing antibodies and the like at present. ByTherefore, the design of S protein-targeted drugs and vaccines becomes the most important direction for the development of SARS-CoV-2 preventive and therapeutic drugs, and the peptide segment (skeleton) of the specific part of S protein polypeptide chain, especially the key peptide segment targeting RBD part, is the most main target for the development of current vaccine design, neutralizing antibodies and other drugs.

However, the S protein of SARS-CoV-2 is a highly glycosylated glycoprotein, each S protein monomer contains at least 22N-linked sugar chains, the S protein of trimer has at least 66 glycosylation sites, and a large number of oligosaccharide-mannose type, high-mannose type, and mannose-containing complex type sugar chains (Zhang, Y) are attached to the surface. et al. Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins. Molecular & cellular proteomics : MCP20, 100058). This means that the new coronavirus is a genuine "sugar-coated virus" and so many glycosylation sites are linked to numerous polysaccharide chains, as if the surface were covered with a thick "sugar chain barrier" or "sugar shield" (glycon shield). This layer is in a dynamic, floating and variable "sugar chain barrier" covering more than 90% of the virus surface (Casalino, L. et al. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein. ACS central science6, 1722-1734) may present difficulties in the development of vaccines and targeted drugs. Antibodies produced by targeted drugs or injected vaccines often have difficulty passing through the surface layer coated with a thick coating to enter and bind internally to the target peptide backbone. Meanwhile, the "floating" sugar chain may shield or even hide the binding site of the antibody or drug, so that the antibody or drug cannot recognize the target site and cannot exert its effect, thereby causing immune escape and drug off-target. Researches show that sugar chains of glycosylation sites N165 and N234 of the S protein can stabilize the open conformation of the S protein, so that the RBD is promoted to be combined with ACE 2; furthermore, the sugar chains of N165, 234, 343 shield the RBD of the S protein in its closed conformation, thus providing protection (Watanabe, y., allen, j. D., wrapp, d., mcLellan, j. S).& Crispin, M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science369, 330-333). Therefore, sugar chains are not protectedBesides covering the S protein peptide chain, the polypeptide can also promote the combination of viruses and receptor proteins, thereby promoting the occurrence of infection.

The problem of immune escape and drug off-target due to the hyperglycosylation of the S protein is therefore a barrier and challenge to the development of SARS-CoV-2 vaccines and other drugs.

Aiming at the possible bottleneck problem of the research and development of the novel coronavirus, the sugar chain trap and the obstacle which are possibly faced by the current vaccine design which aims at the specific part of the S protein peptide chain framework and the research and development of other targeted drugs are avoided, a novel strategy which takes the sugar chain of the S protein as a target is adopted, and the protein polypeptide compound which is specifically combined with the sugar chain is used for sealing the S protein sugar chain, namely a 'pointian' of the novel coronavirus infected host cell, so that the recognition and combination of the virus and a host cell receptor (ACE 2) are blocked, and the aim of preventing and treating the virus infection is fulfilled. Therefore, blocking the recognition binding of the virus to the host cell by binding the sugar chain of the glycoprotein on the surface of the blocked virus is a possible strategy for effectively blocking and preventing viral infection, and thus blocking infection by targeting the sugar chain may become a new breakthrough in drug development.

Lectins are a class of proteins or glycoproteins of non-immunological origin that specifically recognize and bind pairs of sugars and their complexes (Damme, e.j.m. v., peumans, w.j., barre, a.& Rougé, P. Plant Lectins: A Composite of Several Distinct Families of Structurally and Evolutionary Related Proteins with Diverse Biological Roles. Critical Reviews in Plant Sciences17, 575 to 692), rhizome of Siberian solomonseal lectin (C)Polygonatum cyrtonemaHua, lectin, PCL) is a Lectin (An, J) that has been isolated by the inventors from traditional chinese herbal medicine in our country and specifically recognizes and binds mannose and sialic acid. et al.Anti-HIV I/II activity and molecular cloning of a novel mannose/sialic acid-binding lectin from rhizome of Polygonatum cyrtonema Hua. Acta biochimica et biophysica Sinica38, 70-78）。

The invention tests the S protein binding activity and SRAS-CoV-2 antiviral activity of polygonatum agglutinin (PCL), and finds that the polygonatum agglutinin can be specifically and efficiently combined with S protein sugar chains, can be effectively combined with the peptide chain skeleton of S protein, and shows the double binding characteristics of the sugar chains and the peptide chains.

Antiviral activity experiments show that the polygonatum agglutinin protein can effectively block the infection of novel coronavirus to VERO cells, and the antiviral ability of the agglutinin protein is greatly reduced and even lost after mannose is added to seal mannose binding sites of the polygonatum agglutinin protein. Therefore, the polygonatum agglutinin protein can be used as an active ingredient for preparing the medicine for resisting the novel coronavirus. The invention provides possibility for a novel blocking method for blocking SRAS-CoV-2 invasion infection by using polygonatum agglutinin protein so as to achieve the effect of blocking and preventing virus infection.

Disclosure of Invention

The invention aims to provide a novel blocking method for blocking SRAS-CoV-2 invasive infection by using polygonatum sibiricum lectin protein.

The invention also aims to provide the application of the novel blocking method for blocking SRAS-CoV-2 invasive infection by using polygonatum sibiricum lectin protein.

Detailed description of the preferred embodiments

The purpose of the invention is realized by the following experimental scheme:

1) Obtaining an antiviral activity test substance of polygonatum agglutinin protein:

rhizome of Siberian solomonseal lectin (C)Polygonatum cyrtonema，Preparation of the protein hua, lectin, PCL for short, preparation and detection (An, J) were carried out according to methods prior to the inventors' laboratory. et al. Anti-HIV I/II activity and molecular cloning of a novel mannose/sialic acid-binding lectin from rhizome of Polygonatum cyrtonema Hua. Acta biochimica et biophysica Sinica38, 70-78）。

2) And (3) carrying out virus TCID50 (tissue half infection amount) measurement, and testing the antiviral activity of the polygonatum agglutinin of the protein sample to be tested in the step 1).

The invention has the beneficial effects that: the sealwort raw material and the sealwort lectin protein sample are easy to obtain, and compared with the research and development of a neutralizing antibody, a recombinant vaccine, a small molecule drug and the like, the investment cost and the research and development time are greatly reduced; can be specifically targeted and combined with a plurality of oligomannose types, high mannose types and mannose-containing complex sugar chains on the surface of the coronavirus. The research and development of traditional vaccines and targeted drugs are not required to consider the problems of breaking through a large amount of sugar chains attached to the surface, suppressing the immunogenicity of the virus caused by antigen shielding, avoiding the innate/acquired immune response and the like. Thereby efficiently blocking the glycoprotein carbohydrate chains on the surfaces of the viruses or host cells, blocking the recognition and combination of the viruses and the host cells, and further preventing the infection of the novel coronavirus.

Drawings

FIG. 1, (a) normal cell control group, (b) inoculation SARS-CoV-2 group, (c) carbohydrate binding protein group is added.

FIG. 2 neutralization titration curves for PCL and SARS-CoV-2.

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: separating and purifying natural rhizoma Polygonati agglutinin (PCL)

The separation and purification method of Polygonatum sibiricum lectin is described in literature (Bobrocade, zeng-Zhong-Qui, zhou-hong, purification of Polygonatum sibiricum lectin II and partial property research.Journal of biochemistry, 165-170,1996). The method comprises the following steps:

1. crushing fresh rhizoma Polygonati massive stems with high speed tissue triturator, extracting with normal saline, filtering, centrifuging (4 deg.C, 45000r/min,30 min), adding ammonium sulfate into supernatant until 30% saturation, precipitating overnight, and centrifuging at 4 deg.C, 6000r/min for 30min.

2. Collecting supernatant, adding ammonium sulfate to reach 80% saturation, centrifuging at 4 deg.C of 6000r/min for 30min, collecting precipitate, dissolving in water, dialyzing with water, and freeze drying to obtain crude PCL product.

3. Dissolving the PCL crude product with 0.14M NaCl, performing equilibrium dialysis, centrifuging to remove insoluble substances, subjecting the supernatant to affinity chromatography with porcine thyroglobulin-Sepharose 4B column (2.6 × 40 cm), washing with 0.14M NaCl until the absorbance value of the effluent at 280nm is less than 0.02, eluting with 0.2M acetic acid solution at 20ml/h flow rate, collecting fractions, detecting with rabbit hemagglutination experiment, collecting fractions with agglutination activity, dialyzing, and freeze drying.

4. Dissolving PCL sample obtained by affinity chromatography in 0.02M NaAc buffer solution with pH of 4.4, subjecting to equilibrium dialysis, subjecting to CM-Sepharose flash column (2.6 × 28 CM), washing with NaAc buffer solution, subjecting to linear gradient elution with NaAc buffer solution containing 0.6M NaCl, pressurizing with peristaltic pump at flow rate of 3ml/min, collecting fractions with agglutination activity, and freeze drying.

5. The above lyophilized sample was dissolved and equilibrated with 0.14M NaCl on SephadexG-100 (1.6X 100 cm), followed by molecular sieve chromatographic purification of PCL. The flow rate is 12ml/h, and after ultraviolet and blood coagulation activity detection, the II th activity peak is collected, and after dialysis and desalination, the PCL pure product is obtained by freeze drying.

Example 2: virus infection experiment of Vero cell

1. Cell preparation: 100 mu l of Vero E6 cells are inoculated in a 96-well plate, and the number of the cells in each hole is about 8x103 to 1x104. 97% MEM medium (GIBCO, cat # 12800017, naHCO3 addition 1.5 g/L); 3% fetal bovine serum; 5% CO ₂ (ii) a Culturing at 37 ℃. Cells in each well were plated as a monolayer and were approximately 60% abundant to be inoculated with virus.

2. Preparing diluted virus solution: fold of dilution was determined from approximate titer of virus. Virus stock was diluted in EP tubes to X concentrations (10) in serum-free incubators at double ratios ^-1 ，10 ^-2 …10 ^-10 Etc.).

3. Inoculation: the cell culture plate was removed, the culture solution in the 96-well plate was aspirated by a row gun, the incubation solution was aspirated and gently pipetted once per well, and then the incubation solution was aspirated (this step was intended to remove serum, which interferes with the adsorption of the virus). 100 μ l of virus dilution was added to each well, 3 replicates per concentration, and two additional rows were left without virus addition as negative controls. 37 ℃ and 5% CO ₂ And culturing in an incubator for 3 days. And incubating for 1h in a CO2 incubator at 37 ℃, taking out a 96-well plate to absorb virus liquid, adding 200 mul of culture medium, and continuing to cultivate for 3 days in the incubator.

4. Measurement results

The culture plate was removed and the cells were observed under a microscope for pathological changes, and normal cells are shown in FIG. 1 (a), and infected cells are shown in Table 1 and FIG. 1 (b).

The calculation method comprises the following steps:

1) Spearman-Karber method

LgCCID50 /0.2ml= - （X0 - d/2 + d×∑R1/N1）

X0 = lowest log dilution of all lesions

d = logarithm of dilution factor

N1 = number of wells seeded per dilution

R1 = number of lesion holes

Σ = product sum

LgCCID50 /ml = LgCCID50 /0.2ml +0.7

2) Reed-Muench method

CPE (cytopathic effect) was observed, the dilution factor of the virus causing infection in half of the cell flasks/well was found, and the TCID50 of the virus fluid was calculated according to the formula, see Table 1:

TABLE 1 VERO cell CPE after viral infection

(examples)

Distance ratio = (percentage of illness rate higher than 50% -50%)/(percentage of illness rate higher than 50% -percentage of illness rate lower than 50%)

lgTCID50= distance ratio X difference between log of dilutions + log of dilutions above 50% lesion rate TCID50=10-X/0.1ml

The meaning is: 50% of the cells were diseased by 10X inoculation of 100 μ l of the virus.

3) Criteria for determination

The cell control had no pathology and in the absence of standards, the number of experiments should be increased to reduce errors.

Example 3: neutralization experiment of sealwort agglutinin COVID-19 virus

1. Cell preparation: taking Vero cells in good state 1 day before experiment, digesting with pancreatin, counting, and using up

The cells were diluted in the whole medium and prepared at 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 the plate was placed in a CO2 incubator overnight before infection experimentsWhen the cell state was examined, the cells were washed 2 to 3 times with PBS and 100. Mu.L of the maintenance solution was added to each well.

2. The PCL pure product was diluted to 8. Mu.g/mL with PBS as the initial concentration.

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 PCL samples: taking a virus with known TCID50 titer, diluting the virus with a maintenance solution to a concentration of 100 TCID50/35 mu L (which can be adjusted according to the needs of experiments, and part of experiments are 200 TCID50/35 mu L), then adding a virus dilution solution with a volume of 35 mu L into each PCL sample hole, placing the PCL 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 an incubation mixed solution of the virus and the sample according to the volume of 35 mu L/hole after the incubation is finished, adding the virus and the sample into the cell according to the corresponding hole, and placing the cell in a CO2 incubator for incubation for 2 h at 37 ℃.

5. Adding 120 mu L of virus maintenance solution into each cell culture well, placing the cells in a cell culture box for culturing at 37 ℃ for 3-5 days, and observing cell CPE (see figure 1 (C); plasma/serum neutralization titers, or antibody IC50 values, were calculated and the results are shown in figure 2.

Sequence listing

<110> Sichuan university

<120> effect of Polygonatum sibiricum lectin in blocking invasion and infection of novel coronavirus

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gggaacctcg tcatctacga tcagagcaac cgtgtgattt ggcagaccaa gacgaacggg 240

aaggaggacc attacgtgct ggtgctgcag caagaccgca atgtggtcat ctacggccct 300

gtagtttggg ccacaggctc tggaccggcc gtcggactca cccttattcc gcataacgct 360

actgatattg ttcatgctac accgatgctt aatgag 396

Claims (4)

1. The application of natural plant protein in preparing medicine for resisting coronavirus is characterized by that said protein is derived from plant polygonatum rhizome (sealwort)Polygonatum cyrtonemaHua) Produced sealwort lectin (Polygonatum cyrtonemaHua Lectin，PCL）。

2. The use of claim 1, wherein the amino acid sequence of said polygonatum lectin is SEQ ID NO:1 is shown.

3. The use of claim 1, wherein the nucleotide sequence of said Polygonatum cyrtonema lectin is as set forth in SEQ ID NO:2, respectively.

4. Use according to claim 1, wherein the protein is used to block infection of cells by a novel coronavirus.

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