CN113293178A

CN113293178A - Method for detecting neutralizing antibody based on SARS-CoV-2 pseudovirus

Info

Publication number: CN113293178A
Application number: CN202110401752.9A
Authority: CN
Inventors: 王佑春; 黄维金; 张黎; 聂建辉; 张悦; 吴佳静; 李倩倩
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-08-24

Abstract

The invention relates to a method for detecting a neutralizing antibody based on SARS-CoV-2 pseudovirus. The invention also relates to a recombinant vector, a virus-like particle, and a cell strain. The invention also relates to a method for detecting SARS-CoV-2 virus infectivity and a method for detecting the neutralizing activity of SARS-CoV-2 antibody. The constructed cell strain can be stably and efficiently applied to pseudovirus infectivity detection and antibody neutralization activity detection, and the detection cost is reduced.

Description

Method for detecting neutralizing antibody based on SARS-CoV-2 pseudovirus

Technical Field

The invention relates to the field of genetic engineering and molecular biology, in particular to a method for detecting a neutralizing antibody based on SARS-CoV-2 pseudovirus. The invention also relates to a recombinant vector, a virus-like particle, and a cell strain. The invention also relates to a method for detecting SARS-CoV-2 virus infectivity and a method for detecting the neutralizing activity of SARS-CoV-2 antibody.

Background

Pseudoviruses (pseudoviruses) are infectious virus particles which are formed by wrapping a heterologous nucleic acid carrying a reporter gene by a viral capsid protein or an envelope protein and are similar to euviruses, and because the wrapped nucleic acid does not have the whole nucleic acid sequence for replicating and forming the viruses, the pseudoviruses only have one round of infectivity and are safer to operate. High titers of pseudovirus are an important factor in maintaining experimental stability. The titer of the pseudovirus is improved, besides the modification and optimization of a membrane protein gene sequence, the modification can also be carried out on cells infected by the pseudovirus so as to improve the infection efficiency of the virus.

Furin is a type I transmembrane protein present in all vertebrates and in some invertebrates and consists of 794 amino acids; including the N-terminal signal peptide (signal peptide), propeptide (propeptide), catalytic domain (catalytic domain), conserved P domain, Cys-rich domain, transmembrane domain, and cytoplasmic domain. Furin is capable of cleaving precursor proteins and is essential for maintaining the intracellular environment. In addition, Furin is utilized by many viruses and bacteria, and is closely related to the occurrence of certain diseases. While SARS-CoV-2 has unique 4 amino acid insertion (681-PRRA-684) in S1 (nucleotide position 23619 and 23632), and this insertion (PRRA) provides potential recognition and cleavage site for Furin, so that the SARS-CoV-2S protein cleavage efficiency is obviously raised. SARS-CoV-2 also utilizes angiotensin converting enzyme 2(ACE2) as a receptor to bind to the surface of host cells.

In the method for detecting neutralizing antibodies against SARS-CoV-2 pseudovirus, there is a need for a method for detecting neutralizing antibodies against SARS-CoV-2 pseudovirus, which can improve the infection efficiency of SARS-CoV-2 pseudovirus, reduce the amount of virus used, and is stable, efficient and inexpensive.

Disclosure of Invention

Through a large number of experiments and repeated groping, the inventor constructs a cell strain expressing or over-expressing ACE2 protein and Furin protein. Furthermore, the cell strain is applied to establish a method for detecting SARS-CoV-2 virus infection stably, efficiently and at low cost and a method for detecting the neutralizing activity of SARS-CoV-2 antibody.

Accordingly, in a first aspect, the present invention provides a recombinant vector comprising a nucleotide sequence encoding an ACE2 protein and/or a Furin protein.

In certain embodiments, the ACE2 protein and Furin protein are native mammalian (e.g., human) ACE2 protein and Furin protein.

In certain embodiments, the amino acid sequence of the ACE2 protein is set forth in SEQ ID No. 1. In certain embodiments, the amino acid sequence of the Furin protein is set forth in SEQ ID No. 2.

The vector of the present invention may be a cloning vector or an expression vector. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages, cosmids, and the like. In certain embodiments, the vector is capable of expressing a protein of the invention or a fusion protein as described above in a host cell (e.g., a mammalian cell, e.g., a human cell).

In certain embodiments, the recombinant vector is an expression vector. In certain embodiments, the recombinant vector is constructed from a plasmid. In certain embodiments, the plasmid is selected from the group consisting of pLV, pcdna3.1+, pcag3.1, and pcmv3.1.

In certain embodiments, the recombinant vector further comprises a nucleotide sequence encoding a Furin protein signal peptide and/or a nucleotide sequence encoding an ACE2 protein signal peptide.

In some embodiments, the amino acid sequence of the Furin signal peptide is set forth in SEQ ID No. 3. In certain embodiments, the amino acid sequence of the ACE2 protein signal peptide is set forth in SEQ ID No. 4.

In another aspect, the invention provides a viroid-like particle comprising ACE2 protein and/or Furin protein.

In certain embodiments, the viroid is packaged from a mammalian cell co-transfected with a recombinant vector and a nucleic acid component as described above. In the present invention, the recombinant vector as described above self-assembles to form the outer shell of the viroid. In certain embodiments, the viroid further comprises a nucleic acid component. Such nucleic acid components are, for example, encapsulated within a shell formed by self-assembly of the recombinant vector as previously described.

In certain embodiments, the nucleic acid component is selected from any one or more of:

(a) exogenous DNA, or exogenous DNA encoding a foreign protein or polypeptide, or exogenous DNA encoding RNA;

(b) a vector optionally comprising the exogenous DNA described in (a);

(c) exogenous RNA, or exogenous RNA encoding an exogenous protein or polypeptide;

(d) a vector, optionally comprising the exogenous RNA described in (c).

In certain embodiments, the nucleic acid component is a packaging plasmid.

In another aspect, the invention provides a cell line that expresses or overexpresses ACE2 protein and Furin protein.

In certain embodiments, the cell line is derived from transfection of mammalian cells with viroid particles as described previously.

In certain embodiments, the mammalian cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a liver cell, a tumor cell, a neural cell. In certain embodiments, the cell is a 293T cell.

In certain embodiments, the cell strain has integrated into its genome a nucleotide sequence encoding an ACE2 protein and a Furin protein.

In certain embodiments, the ACE2 protein and Furin protein are native human ACE2 protein and native human Furin protein.

In certain embodiments, the genome of the cell strain further incorporates a nucleotide sequence encoding a Furin protein signal peptide and/or a nucleotide sequence encoding an ACE2 protein signal peptide.

In certain embodiments, the cell strain has a collection number of CCTCC No. C202189.

In another aspect, the present invention provides a method of preparing a cell strain as described above, the method comprising:

(1) co-transfecting a mammalian cell with a first nucleic acid component encoding an ACE2 protein and a second nucleic acid component encoding a Furin protein; optionally, co-transfecting a mammalian cell with a third nucleic acid component and the first and second nucleic acid components to obtain a viroid particle;

(2) transfecting the viroid particle into a mammalian cell;

(3) screening the cells obtained in the step (2), wherein the obtained positive cells are the cell strains.

In certain embodiments, in step (1), the third nucleic acid component comprises one or more genes or elements required for a viroid. In certain embodiments, the one or more genes or elements comprise a gag gene, an env gene, and/or a pol gene. In certain embodiments, in step (3), the cells obtained from the culture are screened by antibiotic or fluorescent labeling.

In certain embodiments, the antibiotic is hygromycin or blasticidin.

In certain embodiments, the first nucleic acid component and the second nucleic acid component are contained in the same vector or different vectors.

In certain embodiments, the first nucleic acid component, the second nucleic acid component, and the third nucleic acid component are contained in the same vector or different vectors.

In another aspect, the invention provides a method of detecting SARS-CoV-2 class virus infectivity, the method comprising contacting a cell strain as described above with SARS-CoV-2 class virus and testing the virus titer.

In certain embodiments, the method comprises contacting and incubating a cell strain as described in 3 above with a SARS-CoV-2-like virus, adding a fluorescence detection reagent, detecting the fluorescence value and calculating the viroid titer.

In another aspect, the invention provides a method of detecting the neutralizing activity of a SARS-CoV-2 antibody, the method comprising contacting a SARS-CoV-2 viroid particle or cell strain with the antibody before, simultaneously with or after contacting the viroid particle or cell strain with the cell strain as described above.

In another aspect, the invention provides a kit comprising one or more components selected from the group consisting of: the recombinant vector as described above, the viroid as described above, the cell strain as described above;

optionally, it further comprises one or more selected from:

(1) a first nucleic acid molecule encoding the recombinant vector of claim 1;

(2) one or more helper nucleic acid molecules containing genes or elements required by the viroid particle. In certain embodiments, the one or more helper nucleic acid molecules comprise a gag gene, an env gene, and/or a pol gene.

In certain embodiments, the kit further comprises reagents for transferring the nucleic acid molecule into a host cell, and/or, a vector for inserting the nucleic acid molecule. In certain embodiments, the host cell is a mammalian (e.g., human) cell. In certain embodiments, the host cell is a 293T cell. Optionally, the kit further comprises a host cell.

In certain embodiments, the one or more helper nucleic acid molecules are contained in the same vector or in different vectors.

In certain embodiments, the one or more helper nucleic acid molecules are contained in the same vector or in a different vector than the first nucleic acid molecule.

In certain embodiments, the kit further comprises a SARS-CoV-2 class virus and/or a transfection reagent.

In certain embodiments, the transfection reagent is selected from the group consisting of: lipofectamine2000, lipofectamine3000, PEI, or any combination thereof.

In certain embodiments, the SARS-CoV-2 viroid and the cell strain are selected from a viroid less than 1200TCID50 and less than 5 x 10, respectively⁴Cell line of/well. In certain embodiments, the SARS-CoV-2 viroid and the cell strain are selected from the group consisting of a viroid of 300TCID50 and 1 x 10, respectively⁴Cell line of/well.

In certain embodiments, the kit is used to detect infectivity of a SARS-CoV-2 virus.

In certain embodiments, the kit is used to detect the neutralizing activity of SARS-CoV-2 antibody.

In another aspect, the invention provides a method of screening for a drug candidate capable of inhibiting SARS-CoV-2 infection of a cell, the method comprising contacting a SARS-CoV-2 viroid or a cell strain with the drug candidate prior to, simultaneously with or after contacting the viroid or cell strain with the cell strain as described above.

In another aspect, the invention provides the use of a recombinant vector as described above, a viroid as described above, a cell strain as described above, a kit as described above for detecting the infectivity of a SARS-CoV-2-like virus or for detecting the neutralizing activity of a SARS-CoV-2 antibody.

In another aspect, the present invention provides a method of preparing a viroid particle as described above, comprising:

(a) transfecting a host cell with:

(1) a first nucleic acid molecule encoding the recombinant vector of claim 1;

(2) one or more helper nucleic acid molecules comprising the genes or elements required to form a viroid (e.g., an HIV or SIV virus-like particle); preferably, the one or more helper nucleic acid molecules comprise a gag gene, a pol gene and/or an env gene; and

(b) culturing the host cell under conditions that allow expression of the protein to produce the viroid.

In certain embodiments, the cell is a mammalian (e.g., human) cell. In certain embodiments, the cell is a 293T cell.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, etc., used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "SARS-CoV-2" is an abbreviation for "Severe acute respiratory syndrome coronavirus 2(Severe acute respiratory syndrome coronavirus 2)" which is old called "novel coronavirus" or "2019-nCov", which belongs to the genus β -coronavirus, being an envelope-containing single-stranded positive-sense RNA virus. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN 908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). Like SARS-CoV, the SARS-CoV-2 Receptor is formed by specific binding of Receptor Binding Domain (RBD) on S protein and angiotensin converting enzyme 2(ACE2) on host cell, then the virus undergoes membrane fusion and cell entry, and S protein plays a crucial role in the process of virus infection of cells.

As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia resulting from SARS-CoV-2 infection, which have the same meaning and are used interchangeably.

As used herein, the terms "wild" or "native" are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide or protein, they mean that the nucleic acid molecule, polypeptide or protein exists in nature, is found in nature, and has not been artificially modified or processed in any way. As used herein, native ACE2 protein refers to a naturally occurring, biologically active ACE2 protein. Similarly, the native Furin protein has the same homology. The amino acid sequence of Spike protein can be readily obtained by those skilled in the art from various public databases (e.g., GenBank database). For example, the amino acid sequence of the native ACE2 protein can be as set forth in SEQ ID NO:1 is shown in the specification; the amino acid sequence of the native Furin protein can be shown as SEQ ID NO:2, respectively.

As used herein, the terms "pseudovirus" and "viroid" have the same meaning and are used interchangeably; it is meant that a virus-like particle, formed by self-assembly of viral proteins, does not encapsulate nucleic acids or encapsulates other nucleic acids, such that the pseudovirus or viroid, while able to infect a host cell, is not capable of autonomous replication. Therefore, it is highly biologically safe compared to the true virus. Pseudoviruses are typically packaged in two parts, a packaging component and an expression component. The packaging component is constructed from the viral (e.g., HIV-1) genome with the genetic information required for packaging, reverse transcription, and integration removed, providing the proteins necessary for pseudovirions; the expression component is complementary to the packaging component, contains the genetic information required for packaging, reverse transcription and integration, and also contains the exogenous gene of interest. The pseudovirions can be harvested in the cell supernatant by co-transfecting the host cells with the packaging component and the vector component.

As used herein, the terms "backbone plasmid" and "packaging plasmid" have the same meaning and are used interchangeably. As is generally understood by those skilled in the art, a viral vector system (particularly a lentiviral vector system) may be composed of two parts, i.e., a packaging component (e.g., a packaging plasmid or backbone plasmid) and a vector component (e.g., a recombinant expression vector carrying a gene of interest); among other things, the packaging components (e.g., packaging plasmid or backbone plasmid) can provide all of the helper proteins necessary for transcription and packaging of genetic material (e.g., RNA) into recombinant pseudoviral particles. Thus, high titer pseudovirions can be produced by: cells are co-transfected with the recombinant expression vector and the packaging plasmid, followed by packaging of the pseudovirus in the cells, followed by secretion of the packaged pseudovirus particles into extracellular medium. Such packaging or backbone plasmids are well known to those skilled in the art, for example, backbone plasmids constructed on the basis of HIV-1: including, but not limited to, pSG3.Δ env (Wei X et al, Antibody neutralization and escape by HIV-1.Nature 422:307-312,2003) and NL4-3.fluc.R-. E- (Connor RI, et al, Vpr is required for the implementation of human immunological virus type-1 in monoclonal medicines.virology 206: 935-944, 1995), as well as pSG3.Δ env.fluc and pSG3.Δ env.vcfluc constructed in the examples of this application.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

One skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like. A vector may be introduced into a host cell to produce transcripts, proteins, or peptides therefrom, including by proteins, fusion proteins, isolated nucleic acid molecules, and the like, as described herein.

As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a prophylactically or therapeutically effective amount of a disease (e.g., rotavirus infection) is an amount effective to prevent, prevent or delay the onset of a disease (e.g., rotavirus infection), or to alleviate, reduce or treat the severity of an existing disease (e.g., a disease caused by rotavirus infection). It is within the ability of those skilled in the art to determine such an effective amount. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the term "neutralizing antibody" refers to an antibody having neutralizing activity. The term "neutralizing activity" means that the antibody or antibody fragment has a functional activity of binding to an antigenic protein on the virus, thereby preventing maturation of virus-infected cells and/or virus progeny and/or release of virus progeny, and the antibody or antibody fragment having neutralizing activity can prevent amplification of the virus, thereby inhibiting or eliminating infection by the virus.

As used herein, the term "expression" refers to the transcription and stable accumulation of a nucleic acid fragment (e.g., DNA, mRNA), which also refers to the translation of mRNA into a polypeptide.

As used herein, the term "overexpression" refers to expression higher than endogenous expression of the same or related gene. A heterologous gene is overexpressed if its expression is higher than the expression of its equivalent endogenous gene.

As used herein, the term "transformation" or "transfection" means the introduction of a "foreign" (i.e., heterologous) gene, DNA or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence to produce the desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. Known transfection methods include Agrobacterium tumefaciens-mediated or Agrobacterium rhizogenes-mediated transformation, calcium phosphate transformation, polybrene transformation, protoplast fusion, electroporation, sonication (e.g., sonoporation), liposome transformation, microinjection, naked DNA, plasmid vectors, viral vectors, gene guns (particle bombardment), silicon carbide WHISKERSTM-mediated transformation, aerosol bundling, or PEG transformation, etc.

Advantageous effects of the invention

Compared with the prior art, the cell strain 293T-ACE2-Furin constructed by the application can improve the infection efficiency of SARS-CoV-2 pseudovirus. Under the same virus infection efficiency, the usage amount of the virus can be reduced while the usage amount of the cell is reduced. Furthermore, a stable, efficient and low-cost pseudovirus infectivity detection method and an antibody neutralization activity detection method are established by applying the cell strain.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows a schematic representation of the recombinant vector pLV-ACE 2.

FIG. 2 shows a schematic diagram of the recombinant vector pLV-Furin.

FIG. 3 shows the qPCR detection of ACE2 in 293T-ACE2-Furin cells and 293T cells.

FIG. 4 shows the qPCR detection of Furin in 293T-ACE2-Furin cells and 293T cells.

FIG. 5 shows fluorescence values (RLU) of pseudovirus-infected cells 293T-ACE2, 293T-Furin and 293T-ACE 2-Furin.

FIG. 6 shows the fluorescence values (RLU) of huh7 and 293T-ACE2-Furin of pseudovirus infected cells.

FIG. 7 shows fluorescence values (RLU) of different concentrations of pseudovirus infected cells 293T-ACE2-Furin and huh7 at different concentrations.

FIG. 8 shows the results of the detection of ID50 for neutralizing antibodies at different cell and virus amounts.

FIG. 9 shows the inhibition rate of cells at different cell and virus amounts.

Description of biological Material preservation

Human kidney epithelial cells 293T-ACE2-Furin were deposited at the China Center for Type Culture Collection (CCTCC) located at the Wuhan university Collection in the Wuchang area of Wuhan university, Hubei, opposite the first subsidiary school of Wuhan university, with a collection number CCTCC No: C202189 for a period of 2021, 4 months and 13 days.

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

TABLE 1 sequence information

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook (Sambrook), friesch (Fritsch), and manitis (manitis), molecular cloning: a LABORATORY Manual (Molecular CLONING: A Laboratory Manual), 2 nd edition (1989); a Current Manual of MOLECULAR BIOLOGY experiments (Current PROTOCOLS IN MOLECULAR BIOLOGY BIOLOGY) (edited by F.M. Otsubel et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzymology) series (academic Press): PCR 2: practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. Mefferson, B.D. Hemsh (B.D. Hames) and G.R. Taylor (edited by G.R. Taylor) (1995)), and animal cell CULTURE (ANIMAL CELL CURTURE) (edited by R.I. Fresherni (R.I. Freshney) (1987)).

In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 construction and validation of 293T-ACE2-Furin cells

As shown in FIG. 1, the nucleotide sequences encoding the signal peptide of ACE2 protein (SEQ ID NO:4), ACE2 protein (SEQ ID NO:1), and FLAG tag (SEQ ID NO:9) were ligated to pLV plasmid (purchased from Carriers under the accession number Ecoli (VB200421-1213bkd)) to construct a recombinant vector pLV-ACE 2. Similarly, as shown in FIG. 2, the nucleotide sequences encoding the Furin protein signal peptide (SEQ ID NO:3), the Furin protein (SEQ ID NO:2) and the MYC tag (SEQ ID NO:10) were ligated to pLV plasmid (purchased from vector house, cat # Ecoli (VB200420-1476wpj)) to construct a recombinant vector pLV-Furin. The recombinant vectors pLV-ACE2 and pLV-Furin are packaged into lentiviruses (ACE2 overexpression lentivirus: purchased from carrier house, Cat number LVS (VB200421-1213bkd) -C; FURIN overexpression lentivirus: purchased from carrier house, Cat number LVS (VB200420-1476wpj) -C), the viruses are collected after 48h and concentrated and purified to determine the virus titer, finally the 293T cells are infected by the purified viruses, and the 293T cells are screened by hygromycin 150ug/ml and blasticidin 15ug/ml until blank control is completely killed, namely cell strains which can stably express ACE2 and Furin 293T (named as human kidney epithelial cells 293T-ACE2-Furin, and hereinafter referred to as 293T-ACE2-Furin cells) are obtained.

And verifying whether the stably transformed cell strain is successful or not by qPCR. The method comprises the following specific steps:

1. the extraction of viral nucleic acids was performed using the QIAampViral RNA Mini Kit (250)52906 Kit, as follows:

1) diluting the virus solution after ultracentrifugation by 20 times with 1 × PBS;

2) sucking 310 mu L of buffer AVE, adding the buffer AVE into a tube filled with 310 mu g of frozen Carrier RNA, preparing a solution with the concentration of 1 mu g/mu L, fully dissolving, dividing the solution into proper equal parts, freezing and storing at-20 ℃, and repeatedly freezing and thawing for no more than 3 times;

3) checking the buffer AVL for precipitation, and if necessary, incubating at 80 deg.C until the precipitate is dissolved;

4) calculating the volume of Buffer AVL-Carrier RNA AVE mixture required by a batch of samples according to the table 2 and a formula, and gently inverting and uniformly mixing for 10 times without vortex to avoid generating foams;

5) pipetting 560. mu.L of the prepared buffer AVL (containing 5.6. mu.L of Carrier RNA AVE) into a 1.5mL EP tube;

6) adding 140 mu L of sample into the tube, mixing uniformly for 15s, and incubating at room temperature for 10 min;

7) instantaneous centrifugation, and liquid drops on the cover are thrown back to the bottom of the pipe;

8) 560. mu.L of absolute ethanol (96-100%) was added, mixed for 15s, centrifuged instantaneously and the droplets on the cap were spun back to the bottom of the tube. (if the sample amount is larger than 140. mu.L, the amount of absolute ethyl alcohol is proportionally increased, the step is to be fully mixed to form a uniform solution so as to ensure the yield);

9) pipetting 630. mu.L into Column (taking care not to touch the edge of the Column), capping, centrifuging at 6000 Xg (8000rpm) for 1min, and placing Column into a clean collection tube;

10) repeating the steps until all the lysate is loaded on the column;

11) add 500. mu.L AW1 (ensure that absolute ethanol has been added), centrifuge at 8000rpm for 1min, place Column in a new collection tube (no increase in AW1 is required even if the sample size is greater than 140. mu.L);

12) add 500. mu.L AW2 (ensure that absolute ethanol has been added), centrifuge for 3min at 14000 rpm;

13) discarding waste liquid, replacing a new collecting pipe, and centrifuging at 14000rpm for 1 min;

14) changing 1.5mL EP tube, adding 30 μ L AVE into Column, standing at room temperature for 1min, and centrifuging at 8000rpm for 1 min. The eluted viral RNA can be stably stored for 1 year at-20 ℃ or-70 ℃.

TABLE 2 Buffer AVL and Carrier RNA AVE mixing volume table

2. The Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (50kit)18080 and 051 kit was used for reverse transcription, and the specific operation was as follows:

1) mixing the reagents evenly and centrifuging before use;

2) mu.L of the extracted viral RNA, 1. mu.L of 50. mu.M oligo (dT), and 1. mu.L of 10mM dNTP were combined to prepare a reaction solution A, which was mixed and reacted at 65 ℃ for 5 min.

3) A solution B for cDNA synthesis was prepared, and as shown in Table 3:

TABLE 3 liquid B composition

4) Mixing solution A and solution B, reacting at 50 deg.C for 50min, and reacting at 85 deg.C for 5 min.

5) After completion, 1. mu.L of RNase H was added and reacted at 37 ℃ for 20 min.

6) Finally, the cDNA was kept at-20 ℃ for further use.

3. Virus quantification was performed by TaKaRa TB Green Premix Ex Taq II (Tli RnaseH Plus) RR820A dye method Real-Time PCR as follows:

establishment of a standard curve:

1) synthesizing upstream and downstream primers, wherein the specific sequences are shown in Table 1 and Table 4:

TABLE 4 sequences of primers

2) The plasmid standard is subjected to gradient dilution (50000 ng/. mu.L-3.2 ng/. mu.L) and is used as a template to be added into a reaction system, and 3 compound wells are arranged, wherein the reaction system is shown in Table 5:

TABLE 5 reaction System

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; PCR reaction at 95 ℃ for 3s and 60 ℃ for 30s, reading fluorescence data, and cycling for 40 cycles. The reaction was subjected to melting curve analysis.

3) From the fluorescence data read, the cycle threshold (Ct) was automatically analyzed by the system software and a standard curve was generated.

Quantitative analysis of a sample to be detected:

1) carrying out quantitative analysis on a sample to be detected according to a reaction system and conditions of a standard curve;

2) after the reaction is finished, the specificity of the PCR product is analyzed according to a melting curve, and a quantitative result is analyzed by an ABI 7500 Fast RT-PCR instrument.

The qPCR results are shown in FIG. 3 and FIG. 4, the expression of ACE2 and Furin of 293T-ACE2-Furin cells is improved by more than 100 times compared with 293T cells.

Example 2 comparison of infectivity of pseudoviruses in different cells

Cell lines over-stably expressing ACE2 and cell lines over-stably expressing Furin were prepared according to the method of example 1 and designated cell 293T-Furin and cell 293T-ACE2, respectively. This example is intended to test the infection efficacy of cell lines 293T-Furin, 293T-ACE2 and 293T-ACE 2-Furin.

The pseudovirus infectivity testing procedure was as follows:

1) edge sealing: 260ul of high-pressure sterilized water is added into 36 holes on the periphery of the 96-hole plate for sealing, so that errors caused by evaporation of culture medium in the edge holes are reduced.

2) Mu.l pseudovirus stock was added to well B2-G2.

3) The remaining 54 wells were filled with 100. mu.l of medium, and wells B10-G10 served as Cell Controls (CC).

4) Thereafter, from well B2-G2, wells B3-G3 to B9-G9 were diluted 3-fold, and finally 50. mu.l of the liquid taken out from well B9-G9 was discarded.

5) 100. mu.l of 293T-Furin, 293T-ACE2 and 293T-ACE2-Furin cell suspensions (1X 10)⁵cells/mL) were added to 60 wells in the middle of a 96-well plate.

6) Place the 96 wells in 5% CO₂And culturing in an incubator at 37 ℃ for 24 hours, and detecting.

7) Discard 100 μ l liquid from each well, add 100 μ l Bright-GloTM fluorescence detection reagent, place for 2min at room temperature in the dark, blow repeatedly, and transfer 150 μ l liquid to the whiteboard.

8) Fluorescence values (RLU) were read using a PerkinElmer engsight multifunctional imaging plate reader.

9) The titer of the pseudovirus was calculated by the Reed-Muench method using 3 times the cell control as cut off value.

The stable cell strain is infected by SARS-CoV-2 pseudovirus (the pseudovirus is constructed by the laboratory, the specific construction process of the pseudovirus and other information refer to the natural protocol Quantification of SARS-CoV-2 neutral antibody by a pseudo-transfected virus-based assay 2020 Nov; 15(11):3699-3715.doi:10.1038/s41596-020-0394-5.Epub 2020 Sep 25.), and the result is shown in FIG. 5 that the fluorescence value of 293T-ACE2-Furin the cell is improved by about 20 times compared with 293T-ACE2 and 293T-Furin the case of infecting the same amount of SARS-CoV-2 pseudovirus.

Example 3 establishment of detection method for neutralizing antibody

This example further explores the infectivity of SARS-CoV-2 pseudovirus in Huh-7 and 293T-ACE2-Furin cells. Pseudoviral infectivity test procedure as described in example 2, wherein the same amounts (1200TCID50 and 2 x 10) were added⁴/well) and 293T-ACE2-Furin cells overexpressing ACE2 and Furin.

The result of the reading by the microplate reader is shown in figure 6, the fluorescence value of 293T-ACE2-Furin is more than 5 times of that of Huh-7, and the fact that 293T-ACE2-Furin cells are selected can remarkably improve the infectivity of viruses is proved.

Further, this example compares the fluorescence values of SARS-CoV-2 pseudovirus at different dilutions on 293T-ACE2-Furin cells and Huh-7 cells, and the pseudovirus infectivity test procedure is as described in example 2. As shown in FIG. 7, we found that when 293T-ACE2-Furin cells were used, only 300TCID50 virus amount and 1 x 10 were added⁴The pseudo virus infection efficiency of the cell population/well was compared to the addition of 1200TCID50 virus populations and 2 x 10 virus populations to Huh-7 cells⁴Fluorescence values for cell mass/well are comparable. That is, when 293T-ACE2-Furin cells were used, pseudoviruses were used in one-fourth of the amount originally used, and cells were used in one-half of the amount originally used.

Next, 293T-ACE2-Furin and Huh-7 were used as effector cells for detection of neutralizing antibodies, respectively, and the effect of different amounts of SARS-CoV-2 pseudovirus and different amounts of cells on detection of neutralizing antibodies was compared. 4 different SARS-CoV-2 antibodies are adopted and named as Sample 1-4 respectively, wherein Sample 1 is RBD protein autoimmunity mouse serum 1, Sample2 is RBD protein autoimmunity mouse serum 2(Sample 1 and Sample2 are mouse serum of the laboratory self-immunization, the specific preparation method and the specific preparation process refer to the instruction of antibody preparation and use, Sample3 is JS016 (monoclonal antibody on the market), and Sample4 is DXP-593 sequencing biological monoclonal antibody. First, 4 antibodies were incubated with SARS-CoV-2 pseudovirus for 1 hour, respectively, and then Huh-7 and 293T-ACE2-Furin cells were added for infection, and the procedure for infectivity testing of the pseudovirus was the same as that described in example 2.

As shown in FIG. 8, the range of the change of Sample 1 ID50 (i.e., 50% inhibitory concentration: 50% pseudovirus inhibition, dilution factor of antibody or serum) was 0.5-1.1, using 293T-ACE2-Furin cells (added 300TCID50 of viral load and 110 ×⁴Cell size of/well) ID50 are Huh-7 cells (viral size plus 1200TCID50 and 2 x 10⁴Cell mass/well) 1.05 times; sample2 with ID50 varying between 0.3 and 0.8, using 293T-ACE2-Furin cells (added to 300TCID50 for viral load and 1 × 10⁴Cell size of/well) ID50 is of Huh-7 (viral size plus 1200TCID50 and 2 x 10⁴Cell mass/well) 0.8 times; sample3 with ID50 varying between 0.35 and 1.1, using 293T-ACE2-Furin cells (added to 300TCID50 for viral load and 1 x 10⁴Cell size of/well) ID50 is of Huh-7 (viral size plus 1200TCID50 and 2 x 10⁴Cell mass/well) 1.1 times; sample4 with ID50 varying between 0.35 and 0.75, 293T-ACE2-Furin cells (added 300TCID50 for viral load and 1 x 10⁴Cell size of/well) ID50 is of Huh-7 (viral size plus 1200TCID50 and 2 x 10⁴Cell mass/well) 0.65 times.

As shown in FIG. 9, using 293T-ACE2-Furin cells, a viral load of 300TCID50 and 1 x 10 was added⁴Inhibition of cell mass by/well fitted curve R²Both are greater than 0.99, and the virus amount of 300TCID50 and 1 x 10 are selected based on the most economical principle of virus addition and cell addition, and comparison with Huh-7 for ID50⁴The 293T-ACE2-Furin cell amount of/well is the optimal choice.

In conclusion, the invention successfully constructs 293T cells over-expressing Furin and ACE2, so that neutralizing antibodies are detectedThe virus usage amount is measured from 1.2 x 10³TCID 50/well was reduced to 0.3 x 10³TCID 50/well, thereby establishing a stable, high-efficiency and low-cost SARS-CoV-2 pseudovirus neutralizing antibody detection method.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Sequence listing

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Claims

1. A recombinant vector comprising a nucleotide sequence encoding ACE2 protein and Furin protein;

preferably, the ACE2 protein and Furin protein are native mammalian (e.g., human) ACE2 protein and Furin protein;

more preferably, the amino acid sequence of the ACE2 protein is shown as SEQ ID NO 1; more preferably, the amino acid sequence of the Furin protein is shown as SEQ ID NO. 2;

preferably, the recombinant vector is an expression vector;

more preferably, the recombinant vector is constructed from a plasmid; further preferably, the plasmid is selected from the group consisting of pLV, pcdna3.1+, pcag3.1 and pcmv3.1;

preferably, the recombinant vector further comprises a nucleotide sequence encoding a Furin protein signal peptide and/or a nucleotide sequence encoding an ACE2 protein signal peptide;

more preferably, the amino acid sequence of the Furin signal peptide is shown in SEQ ID NO. 3; more preferably, the amino acid sequence of the ACE2 protein signal peptide is shown as SEQ ID NO. 4.

2. A viroid comprising ACE2 protein and Furin protein;

preferably, the viroid particle is packaged by cotransfecting a mammalian cell with the recombinant vector of claim 1 and a nucleic acid component;

preferably, the nucleic acid component is selected from any one or more of:

(b) a vector optionally comprising the exogenous DNA described in (a);

(d) a vector, optionally comprising the exogenous RNA described in (c);

preferably, the nucleic acid component is a packaging plasmid.

3. A cell strain, wherein the cell expresses or overexpresses ACE2 protein and Furin protein;

preferably, the mammalian cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a liver cell, a tumor cell, a neural cell; more preferably, the cell is a 293T cell;

preferably, the genome of said cell strain has integrated therein a nucleotide sequence encoding ACE2 protein and Furin protein;

preferably, the ACE2 protein and the Furin protein are natural human ACE2 protein and natural human Furin protein;

preferably, the genome of the cell strain is integrated with a nucleotide sequence coding for a Furin protein signal peptide and/or a nucleotide sequence coding for an ACE2 protein signal peptide;

more preferably, the amino acid sequence of the Furin signal peptide is shown in SEQ ID NO. 3; more preferably, the amino acid sequence of the ACE2 protein signal peptide is shown as SEQ ID NO. 4;

preferably, the preservation number of the cell strain is CCTCC No. C202189.

4. A method of preparing the cell strain of claim 3, the method comprising:

(2) transfecting the viroid particle into a mammalian cell;

(3) screening the cells obtained in the step (2), wherein the obtained positive cells are the cell strains;

preferably, in step (1), the third nucleic acid component contains one or more genes or elements required for a viroid particle; preferably, the one or more genes or elements comprise a gag gene, an env gene and/or a pol gene;

preferably, in step (3), the cells obtained by said culturing are screened by antibiotics or fluorescent labels; more preferably, the antibiotic is hygromycin or blasticidin;

preferably, the first nucleic acid component and the second nucleic acid component are contained in the same vector or in different vectors;

preferably, the first nucleic acid component, the second nucleic acid component and the third nucleic acid component are comprised in the same vector or in different vectors.

5. A method of detecting a SARS-CoV-2 class virus infectivity, the method comprising, contacting the cell strain of claim 3 with a SARS-CoV-2 class virus, and testing the virus titer;

preferably, the method comprises contacting and incubating the cell strain of claim 3 with a SARS-CoV-2-like virus, adding a fluorescence detection reagent, detecting the fluorescence value and calculating the viroid titer.

6. A method of detecting SARS-CoV-2 antibody neutralizing activity, the method comprising contacting a SARS-CoV-2 type viral particle or cell strain with the antibody prior to, simultaneously with, or after contacting the SARS-CoV-2 type viral particle with the cell strain of claim 3.

7. A kit comprising one or more components selected from the group consisting of: the recombinant vector of claim 1, the viroid of claim 2, the cell line of claim 3;

optionally, it further comprises one or more selected from:

(1) a first nucleic acid molecule encoding the recombinant vector of claim 1;

(2) one or more helper nucleic acid molecules containing genes or elements required by the viroid particle; preferably, the one or more helper nucleic acid molecules comprise a gag gene, an env gene and/or a pol gene;

preferably, the kit further comprises reagents for transferring the nucleic acid molecule into a host cell, and/or, a vector for inserting the nucleic acid molecule; preferably, the host cell is a mammalian (e.g., human) cell; preferably, the host cell is a 293T cell; optionally, the kit further comprises a host cell;

preferably, the one or more helper nucleic acid molecules are comprised in the same vector or in different vectors;

preferably, the one or more helper nucleic acid molecules are comprised in the same vector or in a different vector than the first nucleic acid molecule;

preferably, the kit further comprises a SARS-CoV-2 class virus and/or a transfection reagent;

preferably, the transfection reagent is selected from: lipofectamine2000, lipofectamine3000, PEI, or any combination thereof;

preferably, the selection amount of the SARS-CoV-2 virus and the cell strain is less than 1200TCIViroid of D50 and less than 5 x 10⁴Cell lines of/well; more preferably, the SARS-CoV-2 viroid and the cell strain are selected from the viroid and 1 x 10 of 300TCID50 respectively⁴Cell lines of/well;

preferably, the kit is used for detecting the infectivity of SARS-CoV-2 virus;

preferably, the kit is used to detect the neutralizing activity of SARS-CoV-2 antibody.

8. A method of screening for a drug candidate capable of inhibiting SARS-CoV-2 infection of a cell, the method comprising contacting a SARS-CoV-2 viroid particle or cell strain with the drug candidate prior to, simultaneously with, or after contacting the viroid particle with the cell strain of claim 3.

9. Use of the recombinant vector of claim 1, the viroid particle of claim 2, the cell strain of claim 3, the kit of claim 7 for detecting infectivity of SARS-CoV-2-like virus or for detecting neutralizing activity of SARS-CoV-2 antibody.

10. A method of making the viroid particle of claim 2, comprising:

(a) transfecting a host cell with:

(1) a first nucleic acid molecule encoding the recombinant vector of claim 1;

(b) culturing the host cell under conditions that allow expression of the protein to produce a viroid;

preferably, the cell is a mammalian (e.g., human) cell; preferably, the cell is a 293T cell.