CN116514931A

CN116514931A - Preparation method and application of novel multi-fluorescence-development coronavirus pseudovirus

Info

Publication number: CN116514931A
Application number: CN202310062140.0A
Authority: CN
Inventors: 秦晓峰; 潘龙玉; 谭贤魁; 陶柳性; 陈正亮
Original assignee: Ruifengkang Biomedical Technology Zhejiang Co ltd
Current assignee: Ruifengkang Biomedical Technology Zhejiang Co ltd
Priority date: 2022-01-30
Filing date: 2023-01-17
Publication date: 2023-08-01

Abstract

The present disclosure relates to a method for preparing novel coronavirus pseudoviruses with multiple fluorescent color development and uses thereof. In particular, the present disclosure relates to a method for preparing a novel multi-fluorescent-chromogenic coronavirus pseudovirus composition, the novel multi-fluorescent-chromogenic coronavirus pseudovirus composition prepared by the method, and uses thereof. The method can detect the neutralizing antibodies aiming at two or more different novel coronavirus strains at the same time, is closer to the real condition after vaccination, has less detection sample consumption, simultaneously detects two or more indexes of one sample, can save time and sample, improves detection flux and analysis efficiency, can freely match the combination of different novel coronavirus strains, and accurately detects the neutralizing antibody titer and activity titer aiming at different variant viruses. The detection method adopted by the method has the advantages of few operation steps, short detection period, large flux, strong result comparability, high accuracy and good repeatability, and can be standardized and implemented in large scale.

Description

Preparation method and application of novel multi-fluorescence-development coronavirus pseudovirus

Technical Field

The present disclosure relates to the technical field of genetic engineering, and in particular relates to a preparation method of a novel coronavirus pseudovirus with multiple fluorescent color development and a method for simultaneously detecting neutralizing antibodies against a plurality of different novel coronavirus strains by using the same.

Background

World health organizations divide into three classes, namely variants of interest (VOCs), variants of interest (VOIs) and variants under surveillance (VUM) based on the infectivity of the variant, disease severity, risk of reinfection, and impact on diagnostic and vaccine performance.

Wherein the variant of interest (VOC) has the following characteristics: certain genetic sequence changes can affect viral characteristics such as infectivity, disease severity, immune escape, diagnostic or therapeutic escape; epidemic in multiple countries, with increasing numbers of cases over time, global public health is facing new risks, which have been determined to lead to serious community transmission or group infection, etc.; has the trend of continuously enhancing infectivity and toxicity or the trend of reducing effectiveness of means such as public health, social measures, vaccines, treatment methods and the like. By 2021, 12 months and 30 days, there are currently only 5 definitions that meet the variety of interest: alpha, beta, gamma, delta and Omicron.

D614G mutation is the first identified and valued variant, and the transmissibility of the virus is enhanced, and the b.1 lineage based on D614G mutation has become the most widely prevalent variant worldwide. Subsequent appearance of highly infectious, highly pathogenic Alpha (b.1.1.7) has led to the observation that the N501Y mutation alters the structure of RBD etc. on S protein and thus changes clinical characteristics. The same variation also occurs in Beta (B.1.351) and Gamma (P.1), and besides D614G and N501Y, the two variants also carry E484K and K417T/N mutations, so that the affinity of the virus RBD to human ACE2 receptor and the immune escape capacity of the virus are increased. Delta (B.1.617.2) variant has both L452R, T478K and P681R mutations, and is the most prevalent variant worldwide due to enhanced infectivity, strong pathogenicity and immune escape.

Omicron, a new strain, was first identified in south Africa on day 11 and 9 of 2021 and was numbered B.1.1.529. The newly discovered amikacin strain has a plurality of variations, namely 32 variations on the spike protein on the surface, and the novel coronavirus infects human bodies through the binding of the spike protein and a human cell receptor. This new strain "appears to have mutations at all identified antigenic sites," which may affect the recognition of spike proteins by most antibodies, suggesting that it may lead to enhanced infectivity, and resistance to various therapeutic monoclonal antibodies (mAbs), as well as to antibody production induced by existing vaccines. The world health organization has indicated in the latest reports that the novel coronavirus variant strain of the virus obmidkon is "very high" at global overall risk level.

The novel coronavirus (2019-nCov) includes four major structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), envelope protein (E protein). Wherein the S protein is the main protein which binds to the cell surface receptor and completes the infection process when the virus infects the host cell. The S protein plays a key role in protective humoral immunity and cellular immunity induced by SARS coronavirus infection. Thus, the S protein is considered as the most attractive target for SARS-CoV vaccine and therapeutic development. In the face of novel coronaviruses, most of inactivated vaccines, recombinant subunit vaccines, RNA vaccines, viral vector vaccines, monoclonal neutralizing antibodies, fusion inhibitors and the like are designed aiming at S proteins.

At present, the global epidemic prevention situation is still severe, the continuous appearance of variant strains brings great uncertainty, and a reliable detection method is needed to provide technical support for upstream etiology basic research or vaccine and drug development and downstream vaccine immunity effect dynamic monitoring. Currently, virus Neutralization (VNT) and pseudovirus neutralization (pVNT) are the main methods of evaluation of neutralizing antibodies after injection of novel coronavirus vaccines. The virus neutralization method is limited to be performed in a laboratory with more than BSL-3, and the problems of high personnel requirement and small detection flux make the method difficult to be a rapid and efficient detection method.

Vesicular Stomatitis Virus (VSV) is a negative strand RNA virus that infects most mammalian cells and expresses up to 60% of the total protein of the viral protein in the infected cells. In nature, VSV infects pigs, cattle and horses and causes varicella disease near the mouth and feet. Although it has been reported that humans infect VSV, VSV does not cause any serious symptoms in humans. VSV encodes 5 proteins including nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), surface glycoprotein (G) and RNA-dependent RNA polymerase (L). Blocking host cell protein synthesis by VSV matrix protein (M) induces cell death.

Pseudoviruses are a type of chimeric virus particles that utilize viral vector gene editing and recombination techniques to express and assemble another viral envelope protein (envelope protein) on the surface of replication-defective viruses. The pseudovirus has been widely used in vaccine research, antibody neutralization activity potency detection and analysis, simulated virus infection cell research, antiviral drug screening, detection kit reference and quality control product development, etc. because of its unique biosafety, stability and unified standard operation and application. The detection of neutralizing antibodies by the pseudovirus method has more obvious advantages in clinical and industrial research fields with higher flux requirements, such as antiviral inhibitor screening, antibody serology test, vaccine immunogenicity and antibody neutralization efficacy evaluation.

Currently, the detection of neutralizing antibodies by a pseudo-virus method is mainly realized by a chemiluminescent detector, a flow cytometer, a microplate fluorescence cell imager (such as Tecan Spark Cyto, bioTek Cystation 5 and the like). The flow cytometry detection has high resolution, and can identify various fluorescent pseudoviruses, but high-flux test cannot be realized due to the speed limitation of the instrument and the detection method. The method has a plurality of operation steps, and only one novel neutralizing antibody of the coronavirus pseudovirus can be detected at a time, so that the simultaneous detection of the neutralizing antibodies of a plurality of novel coronavirus strains can not be realized. Therefore, the flow cytometry and the chemiluminescent instrument have huge workload for detecting various neutralizing antibodies on clinical samples with large sample volumes, and cannot be applied to large-scale vaccine clinical test samples, serum epidemiological investigation and the like.

Disclosure of Invention

Problems to be solved by the invention

The present disclosure provides a method for preparing multiple fluorescent color development novel coronavirus pseudoviruses and a method for detecting neutralizing antibodies of multiple different novel coronavirus strains by using the same, wherein means such as fluorescence reporter gene screening, pseudovirus skeleton plasmid transformation, reporter gene expression level improvement, combination of expressing different fluorescence reporter gene pseudoviruses, fluorescence reporter gene signal reading mode optimization, mutual interference between multiple fluorescence signals avoidance, and the like are provided, and the method is combined with a multicolor micropore plate fluorescence cell imager to solve bottleneck problems of simultaneously and specifically detecting neutralizing antibody activity titer or titer of multiple different novel coronavirus strains, so that high throughput, standardized and large-scale novel coronavirus neutralizing antibody detection is possible.

Solution for solving the problem

The present disclosure describes the following technical solutions:

(1) A method for preparing a novel multi-fluorescent-chromogenic coronavirus pseudovirus composition, wherein the method comprises the following steps:

(A) Preparing a dVSV delta G pseudovirus group and a recombinant plasmid group, wherein the dVSV delta G pseudovirus group comprises dVSV delta G pseudoviruses respectively expressing different fluorescent proteins, and the recombinant plasmid group comprises recombinant plasmids respectively expressing different novel coronavirus strain S proteins;

(B) Mixing dVSV delta G pseudovirus expressing any fluorescent protein in the dVSV delta G pseudovirus group with recombinant plasmid expressing any novel coronavirus strain S protein in the recombinant plasmid group, and co-transfecting host cells to obtain novel coronavirus pseudovirus expressing fluorescent protein and assembled with the novel coronavirus strain S protein;

(C) Mixing the novel coronavirus pseudoviruses which are prepared in the step (B), respectively express different fluorescent proteins and are assembled with the novel coronavirus strain S protein, so as to obtain the novel multi-fluorescent chromogenic coronavirus pseudovirus composition;

wherein, the gene sequence of the dVSV delta G pseudovirus is shown in SEQ ID NO: shown at 9.

(2) The production method according to (1), wherein each novel coronavirus pseudovirus in the multiplex fluorescent chromogenic novel coronavirus pseudovirus composition correspondingly expresses a fluorescent protein and a novel coronavirus strain S protein, and any two novel coronavirus pseudoviruses express fluorescent proteins different from the novel coronavirus strain S protein;

in a specific embodiment, the multiplex fluorescent chromogenic novel coronavirus pseudovirus composition comprises at least two novel coronavirus pseudoviruses, wherein the at least two novel coronavirus pseudoviruses correspondingly express at least two fluorescent proteins, and the at least two novel coronavirus pseudoviruses correspondingly express at least two novel coronavirus strain S proteins.

(3) The production method according to (1) or (2), wherein the method for producing a dVSV.DELTA.G pseudovirus expressing a different fluorescent protein comprises the steps of:

(A-1) vector construction of dVSV.DELTA.G expressing different fluorescent proteins

Inserting genes expressing fluorescent proteins into gene sequences of the dVSV delta G pseudoviruses, transforming the gene sequences into competent cells for culturing, and recovering plasmids of the dVSV delta G pseudoviruses to obtain dVSV delta G pseudovirus vectors in which genes expressing different fluorescent proteins are inserted; the dVSV delta G pseudovirus vectors expressing different fluorescent proteins are respectively obtained through the steps;

(A-2) preparation of dVSV.DELTA.G pseudoviruses expressing different fluorescent proteins

After host cells are infected with recombinant poxviruses expressing T7 polymerase, respectively diluting the dVSV delta G pseudovirus vectors expressing different fluorescent proteins and helper plasmids pP, pN, pL and pVSVG plasmids expressing virus structural proteins, mixing Lipofectamine LTX with helper plasmids pP, pN, pL and pVSVG plasmid mixed solution, so that the dVSV delta G pseudoviruses are packaged, and then secreted outside the host cells to obtain the dVSV delta G pseudoviruses expressing different fluorescent proteins;

wherein, the nucleotide sequence of the pVSVG plasmid is shown as SEQ ID NO: shown at 10.

(4) The method of producing according to any one of (1) to (3), wherein the method of expressing the recombinant plasmid of the S protein of the different novel coronavirus strain comprises the steps of:

(A-3) Synthesis of S protein Gene of expression novel coronavirus strain

Synthesizing different novel coronavirus strain S protein nucleotide sequences according to the amino acid sequences of the different novel coronavirus strain S proteins, and optimizing codons synthesizing the different novel coronavirus strain S protein nucleotide sequences so that the nucleotides can be efficiently expressed in host cells;

In a specific embodiment, the novel coronavirus strain is a wild-type, alpha, beta, gamma, delta, omicron or other variant strain and subtypes thereof;

(A-4) construction and amplification of plasmid expressing novel coronavirus strain S protein

Amplifying a target fragment of a novel coronavirus strain S protein gene with a deleted C-terminal part, adding a gene encoding a tag at the C-terminal of the fragment to obtain an added gene sequence, inserting the added gene sequence into a plasmid, and amplifying to prepare the novel coronavirus strain S protein gene; the different novel coronavirus strain S proteins are respectively used for obtaining recombinant plasmids for expressing the different novel coronavirus strain S proteins;

in a specific embodiment, in step (A-3), the nucleotide sequence of the S protein is as set forth in SEQ ID NO:11-16, and the nucleotide sequence of the S protein;

in a specific embodiment, in step (a-4), the tag is hemagglutinin HA, and the amino acid sequence encoded by the added gene sequence is as set forth in SEQ ID NO: 17-22.

(5) The production method according to any one of (1) to (4), wherein in the step (B), the recombinant plasmid expressing any one of the novel coronavirus strain S proteins is transfected into a host cell, and then the dVSV.DELTA.G pseudovirus expressing any one of the fluorescent proteins is added for cotransfection to obtain a pseudovirus expressing the fluorescent protein and assembled with the novel coronavirus strain S protein.

(6) The production method according to any one of (1) to (5), wherein, in the step (C), the novel coronavirus pseudoviruses each expressing a different fluorescent protein and assembled with a novel coronavirus strain S protein are mixed in proportion, and the mixed novel coronavirus pseudoviruses express different fluorescent proteins;

in a specific embodiment, at least two novel coronavirus pseudoviruses that correspondingly express at least two fluorescent proteins are mixed in proportion.

(7) The production method according to any one of (1) to (6), wherein the fluorescent protein is two or more selected from the group consisting of GFP, BFP, CFP, YFP, RFP, mCherry, iRFP;

in a specific embodiment, the fluorescent proteins are GFP and mCherry.

(8) The production method according to any one of (1) to (7), wherein the host cell is one or more selected from the group consisting of HEK293T, HEK293FT, HEK293S, HEK293, vero and BHK-21;

in a specific embodiment, the fluorescent protein gene is inserted between the M gene and the L gene of the dvsvΔg vector.

(9) The novel multi-fluorescent-chromogenic coronavirus pseudovirus composition prepared by the preparation method according to any one of (1) to (8).

(10) A method for detecting the activity titer of neutralizing antibodies or the efficacy of neutralizing antibodies against novel coronaviruses using a multiplex fluorogenic novel coronavirus pseudovirus composition, said method comprising the steps of:

incubating a sample to be detected containing a novel coronavirus neutralizing antibody with the multiplex fluorescent chromogenic novel coronavirus pseudovirus composition prepared in any one of the preparation methods described in (1) to (8) or the composition described in (9) in a microplate;

adding target cells expressing hACE2 for co-culture, and respectively detecting and reading signals of two or more than two different fluorescent proteins infected by different novel coronaviruses in the target cells by using a multicolor micropore plate fluorescent cell imager after co-culture to obtain the number of cells infected by each novel coronavirus strain pseudovirus;

calculating neutralization inhibition ratios for each novel coronavirus strain pseudovirus in a sample to be detected by comparing with a negative control which does not contain the novel coronavirus neutralizing antibodies, and detecting the neutralization inhibition ratio under each dilution gradient by performing multiple gradient dilution on the sample to be detected to obtain half neutralization inhibition dilution (ID 50) or half neutralization inhibition concentration (IC 50) so as to obtain a neutralization antibody activity titer value or a neutralization antibody titer value;

In a specific embodiment, the sample to be tested is serum or monoclonal antibodies;

in a specific embodiment, the target cell expressing hACE2 is a BHK-21 cell expressing hACE2, a HEK293T cell expressing hACE2, a BHK-21-hACE2 cell or a HEK293T-hACE2 cell.

ADVANTAGEOUS EFFECTS OF INVENTION

In a specific embodiment, the detection method adopted by the present disclosure can detect neutralizing antibodies against two or more different novel coronavirus strains simultaneously, more closely to detect the actual condition after vaccination.

In a specific embodiment, the detection method adopted by the disclosure has the advantages of less detection sample consumption, and simultaneously detecting two or more indexes of one sample, so that time and samples can be saved, and the detection flux and analysis efficiency can be improved.

In a specific embodiment, the detection method adopted by the disclosure can be freely matched with the combination of different novel coronavirus strains, and the neutralizing antibody titer and the activity titer aiming at different variant viruses can be accurately detected.

In a specific embodiment, the detection method adopted by the present disclosure has the advantages of few operation steps, short detection period, large flux, strong result comparability, high accuracy, good repeatability, and standardized and large-scale implementation.

Drawings

FIG. 1 shows a dVSV.DELTA.G-EGFP plasmid map constructed in accordance with the present disclosure;

FIG. 2 shows a dVSV ΔG-mCherry plasmid map constructed according to the present disclosure;

FIG. 3 shows a dVSV ΔG-BFP plasmid map constructed of the present disclosure;

FIG. 4 shows gel electrophoresis charts of dVSV ΔG-EGFP, dVSV ΔG-mCherry and dVSV ΔG-BFP expression plasmids constructed according to the present disclosure;

FIG. 5 shows a comparison of fluorescent signals of dVSV ΔG pseudoviruses expressing different fluorescent proteins constructed in the present disclosure on a Tecan Spark Cyto multicolor microplate fluorescent cell imager;

FIG. 6 shows a signal plot in different fluorescent channels after mixing three dVSV ΔG pseudoviruses expressing different fluorescent proteins of the present disclosure;

FIG. 7 shows construction and restriction verification of S protein expression plasmids of different novel coronavirus strains of the present disclosure;

FIG. 8 shows Western Blotting verification results of S protein expression of different novel coronavirus strains constructed in the present disclosure;

FIG. 9 shows a graph of fluorescent signals of pseudovirus infection of target cells by different novel coronavirus strains constructed in accordance with the present disclosure;

FIG. 10 shows a comparative bar graph of titers of different novel coronavirus strains constructed in accordance with the present disclosure expressing different fluorescent pseudoviruses;

FIG. 11 shows a bar graph of the present disclosure verifying the comparative agreement of the single fluorescent color development and multiple fluorescent color development of novel coronavirus pseudoviruses against novel coronavirus WT and Delta neutralizing antibody ID50 values in test samples;

FIG. 12 shows a graph of the present disclosure verifying single fluorescent color development versus multiple fluorescent color development of novel coronavirus pseudoviruses versus novel coronavirus WT and Omicron neutralizing antibody ID50 values in test samples.

Detailed Description

Definition of the definition

In the claims and/or the specification of the present disclosure, the words "a" or "an" or "the" may mean "one" but may also mean "one or more", "at least one", and "one or more".

As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps. In the meantime, "comprising," "having," "including," or "containing" may also mean enclosed, excluding additional, unrecited elements or method steps.

Throughout this application, the term "about" means: one value includes the standard deviation of the error of the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as being inclusive of alternatives and "and/or", the term "or" in the claims means "and/or" unless expressly indicated otherwise as being exclusive of each other, as defined by the alternatives or alternatives.

In the present disclosure, the term "pseudovirus" refers to a virus in which one retrovirus has incorporated the envelope glycoprotein of another virus, thereby forming a virus with an exogenous viral envelope, while the genome retains the genomic properties of the retrovirus itself.

In the present disclosure, the term "GFP" refers to green fluorescent protein consisting of 238 amino acids having a molecular weight of about 27kDa; is isolated from jellyfish Aequorea Victoria. GFP can convert blue fluorescence from aequorin by chemical action into green fluorescence by energy transfer. GFP has an excitation wavelength of 488nm and an emission peak at about 507 nm.

In the present disclosure, the term "EGFP" refers to an enhanced green fluorescent protein, which is a mutant of GFP, mutated from phenylalanine to leucine at amino acid 64 of GFP.

In the present disclosure, the term "mCherry" refers to red fluorescent protein from mushroom coral (mushroom coral).

In the present disclosure, the term "BFP" refers to blue fluorescent protein, the excitation wavelength of BFP being 382nm and the emission wavelength being 448nm.

In the present disclosure, the term "RFP" refers to a red fluorescent protein from sea anemone (discosoma. Sp.).

In the present disclosure, the term "YFP" refers to a yellow fluorescent protein, which can be regarded as a mutant of a green fluorescent protein. The fluorescence is shifted towards the red spectrum relative to the green fluorescent protein, mainly due to the change of threonine at position 203 of the protein to tyrosine. The maximum excitation wavelength is 514nm and the maximum emission wavelength is 527nm.

In the present disclosure, the term "CFP" refers to a cyan fluorescent protein, with an excitation wavelength of 405nm and an emission wavelength of 485nm.

In the present disclosure, the term "iRFP" refers to an infrared fluorescent protein whose excitation and emission wavelengths lie in the near-red region (650-900 nm).

In the present disclosure, the aforementioned "GFP", "EGFP", "mCherry", "BFP", "RFP", "YFP", "CFP", "iRFP" are all commercial products.

The "routine biological methods in the art" in the present disclosure can be referred to the corresponding methods described in the publications such as "the latest molecular biology laboratory method Assembly (Current Protocols in Molecular Biology, wiley publication)", "the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual, cold spring harbor laboratory publication)", and the like.

The meaning of the sequence listing shown in this disclosure is as follows:

SEQ ID NO:1 shows the forward primer sequence of the amplified EGFP gene;

SEQ ID NO:2 shows the reverse primer sequence for amplifying the EGFP gene;

SEQ ID NO:3 shows the forward primer sequence for amplifying the mCherry gene;

SEQ ID NO:4 shows the reverse primer sequence for amplifying the mCherry gene;

SEQ ID NO:5 shows the forward primer sequence for amplifying the novel coronavirus S protein gene;

SEQ ID NO:6 shows the reverse primer sequence for amplifying the novel coronavirus S protein gene;

SEQ ID NO:7 shows the forward primer sequence of the amplified BFP gene;

SEQ ID NO:8 shows the reverse primer sequence of the amplified BFP gene;

SEQ ID NO:9 shows the gene sequence of the dsvΔg pseudovirus;

SEQ ID NO:10 shows the nucleotide sequence of the pVSVG plasmid

SEQ ID NO:11-16 show the nucleotide sequences of the optimized WT strain, alpha, beta, gamma, delta and Omicron mutant S proteins of the novel coronavirus, respectively;

SEQ ID NO:17-22 show the amino acid sequences encoded by the C-terminal addition of the gene encoding the tag to the S-protein gene fragment of the novel coronavirus strain with a deletion of the C-terminal portion (i.e., WT strain, alpha, beta, gamma, delta and Omicron mutant strain), respectively.

Wherein, SEQ ID NO:9 is as follows:

SEQ ID NO:10 is as follows:

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SEQ ID NO:11 is as follows:

SEQ ID NO:12 is as follows:

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SEQ ID NO:13 is as follows:

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SEQ ID NO:14 is as follows:

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SEQ ID NO:15 is as follows:

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SEQ ID NO:16 is as follows:

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SEQ ID NO:17 is as follows:

SEQ ID NO:18 is as follows:

SEQ ID NO:19 is as follows:

SEQ ID NO:20 is as follows:

SEQ ID NO:21 is as follows:

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SEQ ID NO:22 is as follows:

examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The reagents and consumables employed in the present disclosure are as follows: 5*Q5 Reaction Buffer (Biolabs B9027S), Q5 Hot start High-Fidelity DNA polymerase (NEB M0493L), dNTP mix (Biolabs PC 2100), mluI-HF (NEB R3198L), nheI (Takara 1241A), xhoI (Takara 1094A), ecoRI (Takara 1040S), ecoRV (Takara 1042S), gel recovery kit (Takara 9762), T4 DNA Ligase Enzyme (NEB M0202L), in-Fusion HD Cloning Plus (Takara 638911), LB medium (Shanghai A507002), 6-well cell culture plates (Corning 3516), T25 cell culture flasks (Corning 430639), lipofectamine LTX (Invitrogen 15338100), PBS (Hyclone SH 30256.01), DMEM High sugar medium (Gibco C11995500), penicillin/streptomycin double antibody solution (Gibco 15140-122), fetal bovine serum (Gibco 10091-148), 0.22. Mu.m (Millipore filter 033 GP).

Unless otherwise indicated, all reagents and consumables in this disclosure are commercially available.

Example 1: preparation of novel coronavirus pseudoviruses with multiple fluorescent color development

(1) dVSV delta G plasmid construction and amplification for expressing EGFP fluorescent protein

Primers shown in Table 1 were designed based on the EGFP gene sequence.

TABLE 1

The plasmid containing the codon optimized EGFP gene is amplified through a specific primer (so that the EGFP gene can be efficiently expressed in a host cell), and an EGFP gene fragment with MluI and NheI enzyme cutting sites at two ends is obtained. Amplification system: 5 XQ 5 Reaction buffer 10. Mu.l, Q5 Hot Start High-Fidelity DNA polymerase 0.5. Mu.l, dNTP mix 4. Mu.l, forward and reverse primers 1.25. Mu.l each, template 1. Mu.l, ddH were synthesized artificially ₂ O32. Mu.l, constituting 50. Mu.l of a reaction system;

optimized amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 64℃for 30sec, elongation at 72℃for 30sec, 30 cycles; finally, the mixture is extended for 10min at 72 ℃;

the amplification product and plasmid dVSV.DELTA.G were digested with MluI and NheI restriction enzymes, respectively, and digested in a 37℃water bath for 2h. The cleavage system is shown in Table 2.

TABLE 2

50 μl system	Volume of
		MluI endonuclease	1.5μl
NheI	1.5μl
		10×H buffer	4μl
Amplification product/dVSV.DELTA.G plasmid	30μl(3μg)
		ddH ₂ O	Added to a total of 50. Mu.l

After the cleavage, the cleavage product of the amplified product and the cleavage product of the dVSV.DELTA.G plasmid were obtained by recovering the amplified product using a Takara gel recovery kit and eluting the amplified product with 30. Mu.l of TE buffer.

The cleavage products were subjected to ligation reaction by T4 ligase at 16℃for 4 hours, and the ligation system was as shown in Table 3.

TABLE 3 Table 3

Component (A)	Volume of
		10×TAKARA T4 ligase buffer	1μl
T4 ligase	1μl
		Amplification product cleavage product	1μl
dVSV delta G plasmid cleavage products	1μl
		Sterilizing water	7μl

After being connected for 4 hours at 16 ℃, the materials are transformed, and the materials are subjected to ice bath for 30 minutes; and (3) carrying out heat shock for 45-90s at 42 ℃, carrying out ice bath for 1-2min, adding 1ml of liquid LB, putting into a shaking table at 180rpm at 37 ℃ for shaking for 1h, and then coating the plates. After 24h incubation at 37℃positive clones were picked up on plates, placed into 6ml of inoculum and subjected to shaking at 180rpm at 37℃overnight. Then, plasmid enzyme digestion identification is carried out after plasmid small extraction. After the identification is correct, the dVSV delta G-EGFP plasmid for expressing the fluorescent protein can be obtained.

(2) dVSV delta G plasmid construction and amplification for expressing mCherry and BFP fluorescent protein

Primers as shown in table 4 were designed based on mCherry and BFP gene sequences:

TABLE 4 Table 4

mCher optimized for artificially synthesized codons by specific primers, respectivelyThe ry gene and the BFP gene are amplified (so that the mCherry gene and the BFP gene can be efficiently expressed in a host cell) to obtain mCherry gene and BFP gene fragments with XhoI and NheI enzyme cutting sites at two ends. Amplification system: 5 XQ 5 Reaction buffer 10. Mu.l, Q5 Hot Start High-Fidelity DNA polymerase 0.5. Mu.l, dNTP mix 4. Mu.l, forward and reverse primers 1.25. Mu.l each, template 1. Mu.l, ddH were synthesized artificially ₂ O32. Mu.l, constituting 50. Mu.l of a reaction system;

optimized amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 64℃for 30sec, elongation at 72℃for 30sec,30 cycles; finally, the mixture is extended for 10min at 72 ℃;

the amplification products and dVSV.DELTA.G were digested with XhoI and NheI restriction enzymes, respectively, and digested in a water bath at 37℃for 2 hours, and the digestion system was as shown in Table 5.

TABLE 5

50 μl system	Volume of
		XhoI endonuclease	1.5μl
NheI	1.5μl
		10×H buffer	4μl
Amplification product/dVSV.DELTA.G plasmid	30μl(3μg)
		ddH ² O	Added to a total of 50. Mu.l

The cleavage products were subjected to ligation reaction by T4 ligase at 16℃for 4 hours, and the ligation system was as shown in Table 6.

TABLE 6

Component (A)	Volume of
		10×TAKARA T4 ligase buffer	1μl
T4 ligase	1μl
		Amplification product cleavage product	1μl
dVSV delta G plasmid cleavage products	1μl
		Sterilizing water	6μl

After being connected for 4 hours at 16 ℃, the materials are transformed, and the materials are subjected to ice bath for 30 minutes; and (3) carrying out heat shock for 45-90s at 42 ℃, carrying out ice bath for 1-2min, adding 1ml of liquid LB, putting into a shaking table at 180rpm at 37 ℃ for shaking for 1h, and then coating the plates. After 24h incubation at 37℃positive clones were picked up on plates, placed into 6ml of inoculum and subjected to shaking at 180rpm at 37℃overnight. Then, plasmid enzyme digestion identification is carried out after plasmid small extraction. After the identification, dVSV delta G-mCherry and dVSV delta G-BFP plasmids expressing fluorescent proteins can be obtained.

(3) Preparation of dVSV delta G pseudoviruses expressing different fluorescent proteins

HEK293T cells cultured in log phase were plated into 6 well plates 1-2X 10 per well ⁶ Cells, 2ml complete medium.

Cell plating: adding 1-5×10 culture holes for packaging pseudoviruses after plating for 14-16 hr ⁵ After incubation for 1h at 37℃the supernatant in the packaging wells was changed to serum-free medium Opti-MEM.

Cell transfection: the dVSV ΔG-EGFP, dVSV ΔG-mCherry and dVSV ΔG-BFP plasmids were diluted with 200. Mu.l of Opti-MEM medium, respectively, with a total plasmid amount of 2-5. Mu.g (dVSV ΔG-EGFP, dVSV ΔG-mCherry or dVSV ΔG-BFP plasmid plus packaging helper plasmids pP, pN, pL); another 0.1-1 μg of pVSVG was added. 7.5. Mu.l PLUS Reagent was added. Mu.l of Lipofectamine LTX was diluted with 200. Mu.l of medium. 200. Mu.l of LTX dilution was mixed with 200. Mu.l of plasmid dilution and incubated at room temperature for 10min. The culture medium in the 6-hole plate is changed into Opti-MEM culture medium, and then the mixed solution is added into the 6-hole plate for culturing cells drop by drop, and the 6-hole plate is gently shaken to be uniformly distributed in the 6-hole plate. After 6-8h of transfection, the transfection reagent was aspirated and 2ml of fresh complete medium was added.

Wherein, the nucleotide sequence of the pP plasmid is SEQ ID NO in CN110819657A patent: 9; the nucleotide sequence of the pN plasmid is SEQ ID NO in CN110819657A patent: 7; the nucleotide sequence of the pL plasmid is SEQ ID NO in CN110819657A patent: 8, the disclosure of the aforementioned CN110819657a patent is incorporated by reference into the present disclosure.

Harvesting dVSV delta G-EGFP, dVSV delta G-mCherry and dVSV delta G-BFP pseudoviruses after 24-48 hours, filtering by using a 0.22 mu m filter, collecting, and storing the collected viruses in a refrigerator at the temperature of minus 80 ℃ for a long time.

(4) Construction and amplification of S protein plasmid expressing novel coronavirus strain

The sequences of the S protein genes of the novel coronavirus Wild Type (WT), alpha, beta, gamma, delta and Omicron mutant strains were downloaded from NCBI, and codons were optimized to obtain the nucleotide sequences of WT, alpha, beta, gamma, delta and Omicron mutant strain S genes, respectively, so that they could be efficiently expressed in host cells, and the sequences were submitted to Nanjin Style biosynthesis.

Based on the S protein gene sequence, general primers as shown in Table 7 were designed.

TABLE 7

The artificially synthesized Wild Type (WT), alpha, beta, gamma, delta and Omicron mutant S protein genes were amplified by S protein universal primers.

The nucleotide sequence of the S protein gene is shown as SEQ ID NO: 11-16.

Amplification system: 5 XQ 5 Reaction buffer 10. Mu.l, Q5 Hot Start High-Fidelity DNA polymerase 0.5. Mu.l, dNTP mix 4. Mu.l, forward and reverse primers 1.25. Mu.l each, template 1. Mu.l, ddH were synthesized artificially ₂ O32. Mu.l, constituting 50. Mu.l of a reaction system;

Amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 62℃for 30sec, elongation at 72℃for 30sec,30 cycles; finally, the mixture is extended for 10min at 72 ℃;

the expression vector pcDNA3.1 was digested with EcoRI and XhoI restriction enzymes.

The optimized enzyme digestion system: 25. Mu.l of expression vector, 2. Mu.l of restriction enzyme, 5. Mu.l of 10 Xbuffer, ddH ₂ O16. Mu.l, and digested at 37℃for 1-4h.

And (3) purifying and recovering the PCR target product and the pcDNA3.1 linear vector by electrophoresis. And (3) carrying out In-fusion recombination connection on the recovered PCR amplification product S gene and the pcDNA3.1 expression vector.

The optimized connection system: 5 XIn-Fusion HD Enzyme Premix. Mu.l, linearized vector. Mu.l, purified PCR fragment. Mu.l, ddH ₂ O2. Mu.l, 10. Mu.l of the reaction system was constituted, and the reaction system was water-bath at 50℃for 5-20min.

The ligation products were directly transformed into E.coli DH 5. Alpha. Competent bacteria, screened with ampicillin media, and the monoclonal colonies were picked up for extensive cultivation. pcDNA3.1 plasmids expressing different novel coronavirus strains were obtained.

(5) Novel coronavirus pseudovirus packaging experiment for expressing different fluorescent proteins

HEK293T cells were harvested after digestion, plated into 2T 25 flasks, 5ml per flask, 2-4X 10 per flask ⁶ Individual cells.

The medium in the T25 flask was replaced with serum-free Opti-MEM medium, 5ml per flask. A plasmid transfection reagent mix was prepared, and 2-6. Mu.g of pcDNA3.1-WT and pcDNA3.1-Delta plasmids (200. Mu.l Opti-MEM medium) were added, followed by 18. Mu.l PEI (200. Mu.l Opti-MEM medium) and incubated at room temperature for 15min after mixing, and each was added to a cell flask.

After 8h, the supernatant from each well was changed to 5ml of complete medium. Culturing in a 35 ℃ incubator.

After 24h of incubation, the viruses dVSV.DELTA.G-EGFP and dVSV.DELTA.G-mCherry 6-10X 10 were added, respectively ⁶ pfu, after 8h, was washed 2 times with 10ml PBS and replaced with 5ml complete medium.

After 48h of culture, the virus supernatant was harvested, centrifuged at 2000g for 10min, and the pellet was discarded, and the supernatant was WT pseudovirus expressing GFP and Delta pseudovirus expressing mCherry, respectively.

(6) Mixed preparation of novel coronavirus with multiple fluorescent chromogenes

The WT and Delta pseudovirus titers were determined separately, and then the amount of each pseudovirus was taken based on the virus titer to keep the number of different pseudovirus-infected cells expressing different fluorescent proteins at the same level, and mixed to produce a multiplex fluorescent chromogenic pseudovirus.

Example 2: multiplex fluorescent chromogenic novel coronavirus pseudovirus and neutralizing antibody detection effect verification test

(1) Enzyme digestion verification of dVSV delta G-EGFP, dVSV delta G-mCherry and dVSV delta G-BFP expression plasmids

Double digestion verification is carried out on the constructed dVSV delta G-EGFP, dVSV delta G-mCherry and dVSV delta G-BFP expression plasmids by using NheI and XhoI, agarose gel electrophoresis is carried out after digestion is finished, and the result is shown in figure 4, so that expression plasmids expressing different fluorescent proteins are successfully constructed.

(2) Fluorescent signal detection of dVSV delta G pseudoviruses expressing different fluorescent proteins

The same titres of dVSV delta G-EGFP, dVSV delta G-mCherry and dVSV delta G-BFP pseudoviruses were used for titres detection by a polychromatic microplate fluorescence cell imager (Tecan Spark Cyto). As shown in FIG. 5, the dVSV ΔG-BFP pseudovirus fluorescence signal was partially undetected by the fluorescence detector under various instrument setting conditions, and the fluorescence signal detection result was inaccurate. In addition, the pseudovirus titer of BFP expression was lower (resulting from weaker fluorescence) than that of the constructed EGFP and mCherry two fluorescent proteins.

(3) Research on mutual interference of fluorescent signals of dVSV delta G pseudoviruses expressing different fluorescent proteins

The same titer of dVSV delta G-EGFP, dVSV delta G-mCherry and dVSV delta G-BFP pseudoviruses were mixed in equal proportions and fluorescent signals of different channels were detected using a polychromatic microplate fluorescent cell imager (Tecan Spark Cyto). As shown in fig. 6, in the blue channel, a green fluorescent protein signal could be detected, indicating that EGFP protein has an interference phenomenon with BFP protein signal, which cannot be removed by changing and optimizing the instrument settings. In the red channel, no green fluorescent protein signal was detected, and in the green channel, no red fluorescent protein signal was detected, indicating that no interference phenomenon exists between EGFP protein and mCherry protein signal, and the result is shown in fig. 6.

In combination with the dVSV delta G-BFP pseudoviruses, the problems of insufficient detection of fluorescent signals and inaccurate results exist, so that the following multiple fluorescent color development experiments are preferably carried out on the pseudovirus combination with the optimal consistency of the green fluorescent protein signals and the red fluorescent protein signal intensities expressed by the dVSV delta G-EGFP and the dVSV delta G-mCherry.

(4) Enzyme digestion verification of S protein plasmids expressing different novel coronavirus strains

The constructed pcDNA3.1-WT, pcDNA3.1-Alpha, pcDNA3.1-Beta, pcDNA3.1-Gamma, pcDNA3.1-Delta and pcDNA3.1-Omicron expression plasmids were subjected to three-cleavage verification by restriction enzymes EcoRI, ecoRV and XhoI, and agarose gel electrophoresis was performed after the cleavage was completed, as shown in FIG. 7, to obtain a linear fragment of about 5.3kb, an S gene fragment of about 1.7kb, an S gene fragment of about 1.2kb and an S gene fragment of about 0.8kb, and the band sizes were expected. And the cloned fragment was subjected to DNA sequence analysis by Sanger sequencing, and the result confirmed that the insert was indeed the intended novel coronavirus S gene sequence.

(5) Western blotting verification of novel coronavirus S protein expression of different mutant strains

Different novel coronavirus strain S protein plasmids are transfected and expressed on HEK293T cells, and after 24 hours of transfection, the cells are collected and the extracted proteins are lysed for Western blotting analysis. As shown in FIG. 8, the constructed different novel coronavirus S protein expression vectors can normally express the corresponding S protein.

(6) Viral titers of WT and Alpha, beta, gamma, delta and Omicron mutants expressing different fluorescent proteins

The pseudovirus titer expressing different fluorescent protein signals constructed by the invention is detected. The results are shown in FIG. 9, FIG. 10 and Table 8, where the titer values of the different fluorescent pseudoviruses of the novel coronaviruses are all greater than 1X 10 ⁶ TU/ml, and there was no significant difference between pseudoviral titers expressing different fluorescent proteins. The experiment shows that the novel coronavirus pseudovirus constructed by the invention has higher infection capability and can be used for preparing the novel coronavirus pseudovirus with multiple fluorescent color development subsequently.

TABLE 8

(7) Multiple fluorescent color development novel coronavirus pseudovirus detection neutralizing antibody titer

The serum sample to be tested for the novel coronavirus vaccine is diluted step by a ratio of 2 times with the complete culture medium for 7 gradients, and the initial dilution is 1:100. The control group is WT-EGFP and Delta-mCherry two independent pseudoviruses, the experimental group is mixed virus of two novel coronavirus pseudoviruses in equal proportion, and the mixed virus is respectively incubated with the gradient diluted serum sample to be tested for 1h at room temperature. The incubated samples were then infected with HEK293T-hACE2 cells and after 48h incubation, the number of different fluorescent cells expressed per well was read using a multicolor microplate fluorescent cell imager (Tecan Spark Cyto). And calculating the titer of the neutralizing antibodies of the serum to be tested. As shown in FIG. 11, the number of neutralizing antibody titer values obtained by the multiplex fluorescent chromogenic fluorescent novel coronavirus pseudovirus system and the monochromatic fluorescent novel coronavirus pseudovirus system are highly consistent.

In addition, 3 different operators perform parallel operation, two independent pseudoviruses of WT-mCherry and Omicron-RFP are used as a control group, an experimental group is mixed viruses of WT-mCherry and Omicron-RFP in equal proportion, and the mixed viruses are respectively incubated with gradient diluted samples to be tested at room temperature to detect the neutralizing antibody titer of the serum samples. The results are shown in fig. 12 and table 9, the titer values of the neutralizing antibodies obtained by the multiple fluorescent color development fluorescent novel coronavirus pseudovirus system and the monochromatic fluorescent novel coronavirus pseudovirus system are also good in consistency, and three detection results are not obviously different (CV is less than 15%), so that the multiple fluorescent color development novel coronavirus pseudovirus prepared by the method is stable, has no mutual interference and good repeatability, and can be widely used for detecting the neutralizing antibodies of the multiple novel coronaviruses.

TABLE 9

Based on the experimental results, the multiple fluorescent color development novel coronavirus pseudovirus provided by the disclosure has no interference phenomenon after different novel coronavirus pseudoviruses expressing two fluorescent proteins are mixed, and the single-color pseudovirus neutralization detection result and the double-color detection result have high consistency, so that the multiple fluorescent color development novel coronavirus pseudovirus neutralization detection system can be used for detecting serum neutralizing antibodies after inoculating novel coronavirus vaccines, and has important significance for improving the detection flux and efficiency of the neutralizing antibodies.

The foregoing examples have expressed only a few embodiments of the present disclosure, which are described in more detail and detail, but are not to be construed as limiting the scope of the present disclosure. It should be noted that variations and modifications can be made by those skilled in the art without departing from the spirit of the disclosure, which are within the scope of the disclosure. Accordingly, the scope of protection of the present disclosure should be determined by the following claims.

Claims

1. A method for preparing a novel multi-fluorescent-chromogenic coronavirus pseudovirus composition, wherein the method comprises the following steps:

2. The method of claim 1, wherein each novel coronavirus pseudovirus in the multiplex fluorogenic novel coronavirus pseudovirus composition expresses a fluorescent protein corresponding to a novel coronavirus strain S protein, and any two novel coronavirus pseudoviruses express fluorescent proteins different from the novel coronavirus strain S protein;

preferably, the multiple fluorescent chromogenic novel coronavirus pseudovirus composition comprises at least two novel coronavirus pseudoviruses, wherein the at least two novel coronavirus pseudoviruses correspondingly express at least two fluorescent proteins, and the at least two novel coronavirus pseudoviruses correspondingly express at least two novel coronavirus strain S proteins.

3. The preparation method according to claim 1 or 2, wherein the method for preparing the dsvΔg pseudovirus expressing different fluorescent proteins comprises the steps of:

4. A method of preparation according to any one of claims 1-3, wherein the method of expressing recombinant plasmids of different novel coronavirus strain S proteins comprises the steps of:

(A-3) Synthesis of S protein Gene of expression novel coronavirus strain

alternatively, the novel coronavirus strain is a wild-type, alpha, beta, gamma, delta, omicron or other variant virus strain and subtype thereof;

preferably, in step (A-3), the nucleotide sequence of the S protein is shown in SEQ ID NO:11-16, and the nucleotide sequence of the S protein;

Preferably, in the step (a-4), the tag is hemagglutinin HA, and the amino acid sequence encoded by the added gene sequence is as shown in SEQ ID NO: 17-22.

5. The preparation method according to any one of claims 1 to 4, wherein in the step (B), the recombinant plasmid expressing any one of the novel coronavirus strain S proteins is transfected into a host cell, and then the dsvΔg pseudovirus expressing any one of the fluorescent proteins is added for co-transfection to obtain a pseudovirus expressing the fluorescent protein and assembled with the novel coronavirus strain S protein.

6. The production method according to any one of claims 1 to 5, wherein in the step (C), the novel coronavirus pseudoviruses each expressing a different fluorescent protein and assembled with a novel coronavirus strain S protein are mixed in proportion, and the mixed novel coronavirus pseudoviruses express different fluorescent proteins;

preferably, at least two novel coronavirus pseudoviruses which correspondingly express at least two fluorescent proteins are mixed in proportion.

7. The production method according to any one of claims 1 to 6, wherein the fluorescent protein is two or more selected from the group consisting of GFP, BFP, CFP, YFP, RFP, mCherry, iRFP; preferably, the fluorescent proteins are GFP and mCherry.

8. The production method according to any one of claims 1 to 7, wherein the host cell is one or more selected from the group consisting of HEK293T, HEK293FT, HEK293S, HEK293, vero and BHK-21; preferably, the fluorescent protein gene is inserted between the M gene and the L gene of the dvsvΔg vector.

9. The novel multi-fluorescent-chromogenic coronavirus pseudovirus composition prepared by the preparation method according to any one of claims 1 to 8.

10. A method for detecting the active titer value or neutralizing antibody titer value of a novel coronavirus neutralizing antibody using a multiplex fluorescent chromogenic novel coronavirus pseudovirus composition, said method comprising the steps of:

incubating a sample to be detected containing a novel coronavirus neutralizing antibody with a multiplex fluorescent chromogenic novel coronavirus pseudovirus composition prepared in any one of the preparation methods of claims 1 to 8 or a composition of claim 9 in a microplate;

The neutralization inhibition ratio for each novel coronavirus strain pseudovirus in the sample to be detected is calculated by comparing with a negative control not containing the novel coronavirus neutralizing antibody, and the neutralization inhibition ratio under each dilution gradient is detected by subjecting the sample to be detected to double gradient dilution to obtain half neutralization inhibition dilution (ID 50) or half neutralization inhibition concentration (IC 50),

further obtaining the activity titer value or the neutralizing antibody titer value of the novel coronavirus neutralizing antibody;

optionally, the sample to be detected is serum or monoclonal antibody;

alternatively, the target cell expressing hACE2 is a BHK-21 cell expressing hACE2, a HEK293T cell expressing hACE2, a BHK-21-hACE2 cell or a HEK293T-hACE2 cell.