Plasmid combination and pseudovirus for detecting poliovirus neutralizing antibody

Abstract

The invention belongs to the field of biological medicine, and relates to a plasmid combination, a pseudovirus and a pseudovirus combination for detecting a poliovirus neutralizing antibody, in particular to high-throughput detection of the poliovirus neutralizing antibody. Specifically, the invention relates to a plasmid combination, which comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid. The pseudovirus combination disclosed by the invention can be used for simultaneously detecting three types of neutralizing antibodies of spine gray, is high in sensitivity and good in specificity and repeatability, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a plasmid combination, a pseudovirus and a pseudovirus combination for detecting polio virus neutralizing antibodies, especially high-throughput detection of polio virus neutralizing antibodies. The present invention also relates to a method for preparing a pseudovirus, and a method for high-throughput detection of polio virus neutralizing antibodies. Background Art

[0002] Poliomyelitis (Polio, referred to as polio) is an acute intestinal infectious disease caused by poliovirus (PV, referred to as poliovirus), which mainly affects children under the age of five. Poliovirus is mainly transmitted through fecal-oral transmission. After invading the human body, it mainly invades the central nervous system. In severe cases, it can cause irreversible limb paralysis, commonly known as "polio".

[0003] The virus belongs to the Picornaviridae family, Enterovirus genus. The virus has a diameter of 20-30nm, contains single-stranded positive-strand RNA, and has no envelope. The RNA genome of the poliovirus is about 7500nt long, and is mainly divided into three parts: the 5' non-coding region, the polyprotein coding region, and the 3' non-coding region. The polyprotein coding region can be divided into three parts: P1, P2, and P3. The P1 region is the structural protein coding region. The encoded structural protein P1 is a precursor protein that constitutes the viral capsid and can be processed by viral proteases into VP0, VP3, and VP1. Among them, VP1 is the most important antigenic determinant that triggers a neutralization reaction and is the most important antigen in the viral immune response. The P2 and P3 regions encode non-structural proteins that regulate protein hydrolysis and replication.

[0004] Poliovirus can be divided into three serotypes (type 1, type 2 and type 3) according to different antigenicity, and there is no cross-immunity between types. Common strains include: attenuated strains (Sabin type 1, Sabin type 2 and Sabin type 3), wild strains (type 1 Mahoney, type 2 MEF-1, type 3 Saukett), vaccine-associated virus (VAPV) and vaccine-derived poliovirus (VDPV). There is cross-immunity between different strains of the same serotype of poliovirus. The structural protein P1 is not highly similar. Although the non-structural proteins P2 and P3 are somewhat conservative, there are also certain variations. These differences may affect the antigenicity, pathogenicity and immunogenicity of the virus. Polio infections are now sporadic worldwide, and there is no most common strain. Currently, there is no specific drug that can cure polio. The main method of preventing polio is vaccination. There are two main types of polio vaccines used worldwide: oral poliomyelitis vaccine (OPV) and inactivated poliomyelitis vaccine (IPV). OPV uses Sabin strains; IPV uses wild strains, including type 1 Mahoney strain, type 2 MEF-1 strain and type 3 Saukett strain; Sabin-IPV uses Sabin strains, including Sabin type 1, Sabin type 2 and Sabin type 3.

[0005] The detection of poliovirus neutralizing antibodies is of great significance for evaluating the effectiveness of polio vaccination, diagnosing poliovirus infection, monitoring poliovirus transmission, and formulating public health strategies. Traditional poliovirus neutralizing antibody detection methods mainly rely on infectious live viruses [1]. This method has limitations such as cumbersome operation, long time consumption, low throughput, and high biosafety requirements (must be carried out in a designated BSL-3 laboratory). In addition, with the acceleration of the global polio eradication process and the implementation of the poliovirus containment plan, there is an urgent need to develop a high-throughput neutralizing antibody detection method that can replace live viruses.

[0006] Neutralizing antibody detection based on polio pseudovirus is an effective method to replace traditional neutralization tests [2-7]. Compared with traditional detection methods, it has the significant advantages of high safety (conducted in BSL-2 laboratories), simplicity, rapidity, and objective quantification. The existing polio pseudovirus neutralization detection methods use RD cell lines and pseudoviruses carrying chemical reporter genes (such as firefly luciferase reporter genes, Luciferase). Only one type of neutralizing antibody can be detected in one test, and the throughput is relatively low [2-7]. In addition, the chemiluminescence detection steps are relatively cumbersome and require the addition of expensive exogenous substrates.

[0007] Therefore, it is necessary to develop a detection method based on fluorescent pseudovirus that can simultaneously detect neutralizing antibodies of three types of polio viruses. This method has the advantages of high detection throughput, automated detection, sample saving, economy and convenience, and provides technical support for the monitoring of neutralizing antibodies of polio viruses in large populations and the evaluation of the efficacy of polio vaccines. Summary of the invention

[0008] After in-depth research and creative work, the inventors used poliovirus capsid protein expression plasmids, replicon plasmids carrying fluorescent reporter genes, and T7-RNA polymerase expression plasmids to co-transfect HEK 293T cells to prepare polio fluorescent pseudoviruses. By optimizing the selection of fluorescent reporter genes, the transfection ratio of plasmids, and sensitive cell lines, three types of high-titer polio pseudoviruses carrying different fluorescent reporter genes E2-Crimson (abbreviated as E2), eGFP, and DsRed-express (abbreviated as RFP) were prepared. By optimizing and developing parameters such as the amount of sensitive cells added to the neutralization test, the amount of virus added, and the detection time, a high-throughput detection method for polio neutralizing antibodies was established using three types of polio fluorescent pseudoviruses and HEK 293T cells. The following invention is provided:

[0009] One aspect of the present invention relates to a plasmid combination, comprising a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0010] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus P1 gene;

[0011] The poliovirus replicon plasmid sequentially comprises: a promoter, a 5'UTR, a fluorescent reporter gene, a 2A peptide site, a P2 gene of the poliovirus, a P3 gene of the poliovirus, a 3'UTR and Poly A, but does not comprise a P1 gene of the poliovirus.

[0012] Without being bound by theory, since the P1 gene in the complete genome sequence of the poliovirus is replaced with a fluorescent reporter gene, the lack of the P1 gene makes the genome of the polio pseudovirus a defective genome. The polio pseudovirus can only replicate in a single round, which is safer.

[0013] Without being bound by theory, P2 and P3 are functional proteins that have protease cleavage effects and affect functions related to virus assembly.

[0014] Without being bound by theory, the 2A peptide site (such as the 2Apro site) is an enzyme cleavage site for poliovirus and has a self-cleavage function, which can avoid or reduce the impact of the fluorescent reporter gene on the maturation of P2 and P3 and / or the assembly of viral particles.

[0015] Without being limited by theory, the function of the polymerase expression plasmid is mainly to bind to the promoter on the poliovirus replicon plasmid to ensure the normal transcription and translation of the poliovirus replicon plasmid.

[0016] In some embodiments of the present invention, the plasmid combination is characterized by any one or more of the following items (1) to (15) (for example, any 2 items, any 3 items, any 4 items, any 5 items, any 6 items, any 7 items, any 8 items, any 9 items, any 9 items, any 10 items, any 11 items, any 12 items, any 13 items, any 14 items or all 15 items):

[0017] (1) The eukaryotic expression plasmid is independently pcDNA3.1, pcDNA3.2, pcDNA3.3 or pcDNA3.4;

[0018] (2) the promoter is a T7 promoter and the RNA polymerase is a T7 RNA polymerase,

[0019] The promoter is a T3 promoter and the RNA polymerase is a T3 polymerase, or,

[0020] The promoter is an SP6 promoter and the RNA polymerase is an SP6 polymerase;

[0021] (3) The fluorescent reporter gene is one or more selected from eGFP, ZsGreen, YPet, RFP, mOrange, dTomato, mCherry, mKate2 and E2;

[0022] (4) the 2A peptide is one or more selected from 2Apro, P2A, T2A, E2A and F2A;

[0023] (5) the 5'UTR is derived from the poliovirus genome;

[0024] (6) the 3'UTR is derived from the poliovirus genome;

[0025] (7) the 5'UTR comprises the sequence shown in SEQ ID NO:4;

[0026] (8) the 3'UTR comprises the sequence shown in SEQ ID NO:5;

[0027] (9) The promoter comprises the sequence shown in SEQ ID NO:6;

[0028] (10) The Poly A comprises the sequence shown in SEQ ID NO:7;

[0029] (11) the 2A peptide site comprises the sequence shown in SEQ ID NO: 8;

[0030] (12) The poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3;

[0031] (13) The type 1 poliovirus is one or more selected from the group consisting of the type 1 Mahoney strain and Genbank Nos. AY184219 (Sabin 1), AF405671, KC880365 and AY518727,

[0032] The type 2 poliovirus is one or more selected from the group consisting of type 2 MEF-1 strain and Genbank Nos. AY184220 (Sabin2), OP410365, MZ150566 and MG212459.

[0033] The type 3 poliovirus is one or more selected from the group consisting of type 3 Saukett strain and Genbank Nos. AY184221 (Sabin3), OP137321, MG212494 and MG212445;

[0034] (14) The ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1;

[0035] (15) The poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A.

[0036] In some embodiments of the present invention, the plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0037] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the P1 gene of type 1, type 2 or type 3 poliovirus;

[0038] The poliovirus replicon plasmid includes: a promoter, a 5'UTR, a fluorescent reporter gene such as E2, a 2A peptide site, a P2 gene of type 1 poliovirus, a P3 gene of type 1 poliovirus, a 3'UTR and Poly A, and does not include the poliovirus P1 gene.

[0039] In some embodiments of the present invention, the plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0040] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the P1 gene of type 1, type 2 or type 3 poliovirus;

[0041] The poliovirus replicon plasmid includes: a promoter, a 5'UTR, a fluorescent reporter gene such as eGFP, a 2A peptide site, a P2 gene of type 2 poliovirus, a P3 gene of type 2 poliovirus, a 3'UTR and PolyA, and does not include the poliovirus P1 gene.

[0042] In some embodiments of the present invention, the plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0043] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the P1 gene of type 1, type 2 or type 3 poliovirus;

[0044] The poliovirus replicon plasmid includes: a promoter, a 5'UTR, a fluorescent reporter gene such as RFP, a 2A peptide site, a P2 gene of type 3 poliovirus, a P3 gene of type 3 poliovirus, a 3'UTR and PolyA, and does not include the poliovirus P1 gene.

[0045] In some embodiments of the present invention, the plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0046] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus type 1 P1 gene;

[0047] The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR (e.g., from poliovirus type 1), a fluorescent reporter gene such as E2, a 2A peptide site, a P2 gene of poliovirus type 1, a P3 gene of poliovirus type 1, a 3'UTR (e.g., from poliovirus type 1), and Poly A, and does not include the poliovirus P1 gene;

[0048] Preferably, the poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A;

[0049] Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1.

[0050] In some embodiments of the present invention, the plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0051] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus type 2 P1 gene;

[0052] The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR (e.g., from a type 2 poliovirus), a fluorescent reporter gene such as eGFP, a 2A peptide site, a P2 gene of a type 2 poliovirus, a P3 gene of a type 2 poliovirus, a 3'UTR (e.g., from a type 2 poliovirus), and Poly A, and does not include a poliovirus P1 gene;

[0053] Preferably, the poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A;

[0054] Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1.

[0055] In some embodiments of the present invention, the plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0056] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus type 3 P1 gene;

[0057] The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR (e.g., from type 3 poliovirus), a fluorescent reporter gene such as RFP, a 2A peptide site, a P2 gene of type 3 poliovirus, a P3 gene of type 3 poliovirus, a 3'UTR (e.g., from type 3 poliovirus), and Poly A, and does not include the poliovirus P1 gene;

[0058] Preferably, the poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A;

[0059] Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1.

[0060] Another aspect of the present invention relates to a plasmid product, comprising a first plasmid combination, a second plasmid combination and a third plasmid combination, wherein the first plasmid combination, the second plasmid combination and the third plasmid combination are any one of the plasmid combinations of the present invention, wherein:

[0061] The first plasmid combination includes a type 1, type 2 or type 3 poliovirus capsid protein expression plasmid, a type 1 poliovirus replicon plasmid and an RNA polymerase expression plasmid;

[0062] The second plasmid combination includes a type 1, type 2 or type 3 poliovirus capsid protein expression plasmid, a type 2 poliovirus replicon plasmid and an RNA polymerase expression plasmid;

[0063] The third plasmid combination includes a type 1, type 2 or type 3 poliovirus capsid protein expression plasmid, a type 3 poliovirus replicon plasmid and an RNA polymerase expression plasmid;

[0064] The first plasmid combination, the second plasmid combination and the third plasmid combination are independent of each other or mixed together;

[0065] The fluorescent reporter genes in the first plasmid combination, the second plasmid combination and the third plasmid combination are different from each other and can emit different fluorescence;

[0066] Preferably, the type 1 poliovirus is one or more selected from the group consisting of type 1 Mahoney strain and Genbank Nos. AY184219 (Sabin 1), AF405671, KC880365 and AY518727.

[0067] The type 2 poliovirus is one or more selected from the group consisting of type 2 MEF-1 strain and Genbank Nos. AY184220 (Sabin2), OP410365, MZ150566 and MG212459.

[0068] The type 3 poliovirus is one or more selected from the group consisting of type 3 Saukett strain and Genbank Nos. AY184221 (Sabin3), OP137321, MG212494 and MG212445;

[0069] Preferably, none of the poliovirus capsid protein expression plasmids in the three plasmid combinations contains the eGFP-2A sequence.

[0070] In some embodiments of the present invention, the plasmid product, wherein the fluorescent reporter gene is selected from eGFP, RFP and E2;

[0071] Preferably, the fluorescent reporter genes in the first plasmid combination, the second plasmid combination and the third plasmid combination are E2, eGFP and RFP respectively;

[0072] Preferably, E2 comprises the sequence shown in SEQ ID NO: 1;

[0073] Preferably, eGFP comprises the sequence shown in SEQ ID NO: 2;

[0074] Preferably, RFP comprises the sequence shown in SEQ ID NO:3.

[0075] In some embodiments of the present invention, the plasmid product, wherein the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the first plasmid combination, the second plasmid combination and / or the third plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1.

[0076] In some embodiments of the present invention, the plasmid product, wherein:

[0077] (1) The first plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0078] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus type 1 P1 gene;

[0079] The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR, a fluorescent reporter gene such as E2, a 2A peptide site, a P2 gene of type 1 poliovirus, a P3 gene of type 1 poliovirus, a 3'UTR and Poly A, and does not include a poliovirus P1 gene;

[0080] Preferably, the poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A;

[0081] (2) The second plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0082] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus type 2 P1 gene;

[0083] The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR, a fluorescent reporter gene such as eGFP, a 2A peptide site, a P2 gene of type 2 poliovirus, a P3 gene of type 2 poliovirus, a 3'UTR and PolyA, and does not include a poliovirus P1 gene;

[0084] Preferably, the poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A;

[0085] and / or

[0086] (3) The third plasmid combination comprises a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein:

[0087] The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus type 3 P1 gene;

[0088] The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR, a fluorescent reporter gene such as RFP, a 2A peptide site, a P2 gene of type 3 poliovirus, a P3 gene of type 3 poliovirus, a 3'UTR and PolyA, and does not include a poliovirus P1 gene;

[0089] Preferably, the poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A.

[0090] Another aspect of the present invention relates to a method for preparing polio pseudovirus, comprising the steps of transfecting eukaryotic cells with the plasmid combination described in any one of the present invention or the plasmid product described in any one of the present invention;

[0091] Preferably, the eukaryotic cells are selected from one or more of HEK 293T, RD, Hep2, Vero and Huh-7 cells;

[0092] Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1.

[0093] Another aspect of the present invention relates to a polio pseudovirus, which is prepared by the method for preparing a polio pseudovirus described in any one of the present invention.

[0094] In some embodiments of the present invention, the polio pseudovirus is a type 1 polio pseudovirus, a type 2 polio pseudovirus or a type 3 polio pseudovirus prepared by the method for preparing a polio pseudovirus of the present invention.

[0095] In some embodiments of the present invention, the polio pseudovirus is type 1-E2 polio pseudovirus, type 2-eGFP polio pseudovirus or type 3-RFP polio pseudovirus prepared by the method for preparing polio pseudovirus of the present invention.

[0096] In some embodiments of the present invention, the polio pseudovirus is Sabin1-E2 pseudovirus, Sabin2-eGFP pseudovirus or Sabin3-RFP pseudovirus prepared by the method for preparing polio pseudovirus of the present invention.

[0097] In some embodiments of the present invention, the polio pseudovirus is a type 1 Mahoney strain-E2 pseudovirus, a type 2 MEF-1 strain-eGFP pseudovirus or a type 3 Saukett strain-RFP pseudovirus prepared by the method for preparing a polio pseudovirus of the present invention.

[0098] Another aspect of the present invention relates to a polio pseudovirus combination, including type 1 polio pseudovirus, type 2 polio pseudovirus and type 3 polio pseudovirus, which are respectively obtained by transfecting eukaryotic cells with the first plasmid combination, the second plasmid combination and the third plasmid combination in any one of the plasmid products of the present invention.

[0099] The type 1 polio pseudovirus, type 2 polio pseudovirus and type 3 polio pseudovirus are independent of each other or mixed together;

[0100] Preferably, the eukaryotic cells are selected from one or more of HEK 293T, RD, Hep2, Vero and Huh-7 cells;

[0101] Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1;

[0102] Preferably, the polio pseudovirus combination includes type 1-E2 polio pseudovirus, type 2-eGFP polio pseudovirus and type 3-RFP polio pseudovirus prepared by the method for preparing polio pseudovirus of the present invention;

[0103] Preferably, the polio pseudovirus combination includes Sabin1-E2 pseudovirus, Sabin2-eGFP pseudovirus and Sabin3-RFP pseudovirus;

[0104] Preferably, the polio pseudovirus combination includes type 1 Mahoney strain-E2 pseudovirus, type 2 MEF-1 strain-eGFP pseudovirus and type 3 Saukett strain-RFP pseudovirus.

[0105] Another aspect of the present invention relates to a method for detecting polio virus neutralizing antibodies, a method for evaluating the efficacy of polio vaccination, a method for diagnosing polio virus infection, or a method for monitoring polio virus transmission, comprising the following steps:

[0106] (1) contacting or incubating an appropriate amount of the polio pseudovirus described in any one of the present invention or the polio pseudovirus combination described in any one of the present invention with a sample to be tested;

[0107] (2) adding sensitive cells and continuing to culture for a period of time, and detecting the number of fluorescent spots; and

[0108] (3) Calculate ED based on the number of fluorescent spots 50 ;

[0109] Preferably, the method further comprises the following steps:

[0110] (4) The obtained ED 50 Compare with the cut off value;

[0111] Preferably, the method is a non-therapeutic and non-diagnostic method.

[0112] In some embodiments of the present invention, the method is characterized by any one or more of the following items ① to ⑨ (for example, any 2 items, any 3 items, any 4 items, any 5 items, any 6 items, any 7 items, any 8 items or all 9 items):

[0113] ① The sample to be tested in step (1) is a serum sample;

[0114] ② The amount of polio pseudovirus in step (1) is 1000 TCID 50 Up to 3500TCID 50 , preferably 1000TCID 50 Up to 3000TCID 50 , 1000TCID 50 Up to 2500TCID 50 、1000TCID 50 Up to 2000TCID 50 or 1500TCID50 ;

[0115] ③ The incubation in step (1) is at 37° C. for 0.5-3 h, for example, 1-2 h;

[0116] ④ The sensitive cells in step (2) are HEK 293T cells;

[0117] ⑤In step (2), the amount of sensitive cells used is 1500 TCID 50 Polio pseudovirus 5×10 3 Up to 8×10 4 cells / well (96-well plate); preferably 4×10 4 Up to 6×10 4 cells / well (96-well plate);

[0118] ⑥ In step (2), the culture is carried out for 6-72 hours, preferably 12-24 hours;

[0119] ⑦ In step (3), the Reed-Muench method or nonlinear regression is used to calculate ED 50 ;

[0120] ⑧ In step (4), the cut-off value for pseudovirus neutralization detection is 15;

[0121] ⑨The method is for pharmaceutical purposes.

[0122] Cut off value, also known as cutoff value, is a standard value used to determine whether a test result is positive. In medical testing, the cut off value is used to distinguish whether the test result is negative or positive. Results above this value are judged as positive, and results below this value are judged as negative.

[0123] The neutralization test is considered by the WHO as the "gold standard" for evaluating whether vaccine-induced antibodies have a protective effect. There is a correlation between serum antibody levels and protective efficacy, and maintaining neutralizing antibody titers above the threshold is a necessary condition for effective protection. The neutralizing antibody titer for live poliovirus infection is approximately 1:(4-8).

[0124] In the diagnosis of poliomyelitis, the quantitative requirement for neutralizing antibodies against live poliovirus is that the serum neutralizing antibody or specific IgG antibody titer of patients in the recovery period is 4 times higher than that in the acute period.

[0125] Another aspect of the present invention relates to the use of any polio pseudovirus described in the present invention or any polio pseudovirus combination described in the present invention in the preparation of a drug for detecting polio virus neutralizing antibodies, evaluating the effectiveness of polio vaccination, diagnosing polio virus infection or monitoring polio virus transmission.

[0126] In the present invention, the term "polio neutralizing antibody" refers to an antibody that can specifically bind to the polio virus and exert a neutralizing effect, which can block the virus from infecting target cells.

[0127] In the present invention, the term "neutralizing antibody titer" is usually expressed as a ratio, such as 1:4 or 1:8. This expression reflects the maximum dilution that can effectively neutralize pathogens after the sample is diluted. Behind this ratio is a quantitative determination of antibody concentration. For example, 1:4 means that after the serum sample is diluted to a concentration of 1 / 4, it can still effectively neutralize the virus or pathogen.

[0128] In the present invention, unless otherwise specified, the "first" (e.g., the first plasmid combination), "second" (e.g., the second plasmid combination) or "third" (e.g., the third plasmid combination) is merely for reference and does not have a particular order meaning.

[0129] Advantageous Effects of the Invention

[0130] The present invention achieves one or more of the following technical effects:

[0131] (1) The detection method of the present invention has high sensitivity, good specificity and repeatability.

[0132] (2) The present invention has a good correlation with the live virus neutralization test results.

[0133] (3) The present invention can simultaneously detect three types of neutralizing antibodies against polio in one reaction well, and has the advantages of high safety, simplicity and rapidity, sample saving, high detection throughput, low detection cost, and objective quantification. It provides a technical means for large-scale population polio neutralizing antibody monitoring, vaccine research and development evaluation, etc.

[0134] (4) The present invention has good specificity among different viruses.

[0135] (5) The present invention has good specificity among poliovirus types. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Figure 1A : Schematic diagram of capsid protein expression plasmid.

[0137] Figure 1B : Schematic diagram of the replicon plasmid.

[0138] Figure 2 :The fluorescent spot counter was used to detect the titer of pseudoviruses carrying 9 different fluorescent proteins under different bands.

[0139] FIG. 3A to FIG. 3C : Optimizing the selection of fluorescent reporter genes for three types of polio pseudoviruses.

[0140] Figure 4 : Optimize the transfection ratio of plasmids (capsid protein expression plasmid, replicon plasmid and T7-RNA polymerase expression plasmid).

[0141] Figure 5A , screening of sensitive cell lines. Figure 5B , optimize the design of capsid protein expression plasmid. Figure 5C , a combination of screening sensitive cell lines and optimizing the design of capsid protein expression plasmids.

[0142] Figure 6 : Optimize detection time.

[0143] Figure 7 : Optimize the amount of cells added.

[0144] Figure 8 : Optimize the amount of pseudovirus added.

[0145] Fig. 9 : Sensitivity validation of a fluorescent pseudovirus neutralization assay.

[0146] Fig. 10A and Fig. 10B :Specificity verification of fluorescent pseudovirus neutralization detection method. Among them: Fig. 10A Demonstrated inter-type specific detection of polioviruses, Fig. 10B Validation of specificity between different viruses is shown.

[0147] Fig.11 : Reproducibility validation of a fluorescent pseudovirus neutralization assay.

[0148] Fig.12 : Correlation of fluorescent pseudovirus neutralization test results with live virus neutralization test results.

[0149] Some of the sequences involved in the present invention are shown in Table A below.

[0150] Table A: Partial sequences of the present invention

[0151]

[0152]

[0153] DETAILED DESCRIPTION

[0154] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0155] Example 1: Preparation of high titer polio fluorescent pseudovirus

[0156] 1. Plasmid Construction

[0157] The eukaryotic expression vectors used in this study were from Invitrogen's pcDNA3.4 vector and Promega's pGEM-T Easy Vector.

[0158] (1) Construction of capsid protein expression plasmid: The poliovirus P1 gene was codon optimized (optimized species Homo sapiens), a kozak sequence (GCCACC) was added before the start codon ATG and constructed into the pcDNA3.4 vector, named capsid protein expression plasmid ( Figure 1A ). The structural protein P1 provides antigenic capsid protein.

[0159] (2) Construction of replicon plasmid ( Figure 1B ): The 5'UTR of the poliovirus genome, different fluorescent reporter genes (eGFP, ZsGreen, YPet, RFP, mOrange, dTomato, mCherry, mKate2 and E2), P2 gene, P3 gene and 3'UTR were constructed behind the T7 promoter of pGEM-T Easy Vector, and a 2Apro site was introduced behind the fluorescent reporter gene, and Poly(A) was added to the end of the 3'UTR.

[0160] (3) The T7 RNA polymerase gene was constructed into the pcDNA3.4 vector and named as T7 RNA polymerase expression plasmid.

[0161] 2. Preparation of Fluorescent Poliovirus

[0162] (1) Prepare cells: HEK 293T cells, with a confluency of 60%-70%, for subsequent transfection.

[0163] (2) Plasmid transfection: The polio capsid protein expression plasmid, replicon plasmid and T7 RNA polymerase expression plasmid were co-transfected into HEK 293T cells. The plasmid transfection operation was performed according to the instructions of lipo3000 transfection reagent (Invitrogen, L3000015).

[0164] (3) Virus collection: 48 h after transfection, the cells and their culture medium were frozen and thawed twice, centrifuged at 4000 rpm for 30 min to remove cell debris, and the supernatant was filtered through a 0.22 μm filter membrane and stored at -80°C after aliquoting.

[0165] 3. Pseudovirus Titration Test

[0166] (1) Add 100 μl of culture medium to 60 wells B2 to G11 of a 96-well plate.

[0167] (2) Add 50 μl of the prepared polio pseudovirus to 6 wells B2-G2.

[0168] (3) Then, starting from well B2-G2, perform 3-fold dilution in sequence to wells B3-G3 and subsequent wells. Finally, discard the 50 μl mixed liquid taken from wells B10-G10.

[0169] (4) Add 100 μl of HEK 293T cells to the middle 60 wells of a 96-well plate (2×10 4 The edge wells were sealed with 250 μl sterile water.

[0170] (5) The 96-well plate was placed in a 5% CO2, 37°C cell culture incubator for 12 hours and then tested.

[0171] (6) Use a fluorescent spot counter (Cytation5, Biotek) to count the number of fluorescent spots in each well.

[0172] (7) Calculation of TCID of poliovirus pseudovirus using the Reed-Muench method 50 (Half tissue cell infective dose, 50% tissue culture infective dose).

[0173] 4. Optimization of fluorescent reporter genes of different types of polio pseudoviruses

[0174] Screening of fluorescent reporter genes: A fluorescent spot counter (Cytation 5, Biotek) was used to detect the titers of polio pseudoviruses carrying 9 different fluorescent protein reporter genes (eGFP, ZsGreen, YPet, RFP, mOrange, dTomato, mCherry, mKate2 and E2) at (EX469 / 35, EM 525 / 39), (EX 531 / 40, EM 593 / 40) and (EX 628 / 40, EM 685 / 40) bands. The results are shown in the figure. Figure 2As shown. The results showed that YPet, mKate2, and mCherry would interfere in the (EX 469 / 35, EM 525 / 39) band, (EX531 / 40, EM 593 / 40) band, and EX 628 / 40, EM 685 / 40) band, so they were deleted. For the remaining 6 fluorescent reporter genes, eGFP had more fluorescent cell dots compared with ZsGreen in the (EX 469 / 35, EM 525 / 39) band; RFP had more fluorescent cell dots compared with mOrange and dTomato in the (EX 531 / 40, EM 593 / 40) band; and only E2 was not affected by the other two excitation lights and had more fluorescent cell dots in the (EX 628 / 40, EM 685 / 40) band. Therefore, the three fluorescent protein reporter genes, eGFP, RFP, and E2, were selected.

[0175] Optimizing the selection of fluorescent reporter genes for different types of polio pseudoviruses: Determining the optimal fluorescent reporter gene combination for poliovirus types 1, 2, and 3, the inventors used replicon plasmids containing three different fluorescent reporter genes, eGFP, RFP, and E2, to prepare 4 strains of type 1, type 2, and type 3 pseudoviruses, respectively, for a total of 12 strains and 36 combinations. The constructed polio pseudoviruses include the Sabin strain and 9 strains randomly selected from NCBI. The Genbank numbers of type 1 poliovirus are AY184219 (Sabin1), AF405671, KC880365, and AY518727; the Genbank numbers of type 2 poliovirus are AY184220 (Sabin2), OP410365, MZ150566, and MG212459; and the Genbank numbers of type 3 polioviruses are AY184221 (Sabin3), OP137321, MG212494, and MG212445.

[0176] The results are as follows FIG. 3A to FIG. 3C The results showed that when poliovirus type 1, type 2, and type 3 corresponded to the fluorescent reporter genes E2, eGFP, and RFP, respectively, the number of fluorescent spot cells was higher in the selected 4 strains. It was finally determined that the fluorescent reporter genes corresponding to poliovirus type 1, type 2, and type 3 were: type 1-E2, type 2-eGFP, and type 3-RFP.

[0177] 5. Optimize the transfection ratio of the three plasmids

[0178] The capsid protein expression plasmid, replicon plasmid and T7 RNA polymerase expression plasmid were co-transfected into packaging cells to prepare polio fluorescent pseudovirus. The three plasmids were transfected at different transfection ratios: 1:3:1, 3:3:1, 1:2:1, 1:1:1. The effects of different transfection ratios of the three plasmids on the titer of fluorescent pseudovirus were compared. The results are shown in Figure 4 As shown. The results showed that when the transfection ratio of capsid protein expression plasmid, replicon plasmid and T7 RNA polymerase expression plasmid was 3:3:1, the number of fluorescent spots was high. Therefore, the optimal transfection ratio of the three plasmids was determined to be 3:3:1.

[0179] 6. Screening of sensitive cell lines and optimization of P1 protein expression plasmid

[0180] RD, HEK 293T, Hep2, Vero, Huh-7 cells, Sabin1, Sabin2, Sabin3, and wild-type 1 Mahoney (Genbank No.: KU866422), wild-type 2 MEF-1 (Genbank No.: AY238473) and wild-type 3 Saukett pseudovirus (Genbank No.: KP247597) were used to perform pseudovirus titration tests. The specific steps are shown in 3. Pseudovirus titration test above to screen sensitive cell lines for high-throughput detection of polio neutralizing antibodies. The results are shown in Figure 2. Figure 5A As shown, the number of positive fluorescent spots of the six strains of pseudovirus was the largest when HEK 293T cells were used as sensitive cells, so HEK 293T cells were selected as sensitive cells.

[0181] The design strategy of the capsid protein expression plasmid in the existing research is kozak-eGFP-2A-P1. The present invention deletes eGFP-2A to increase the titer of pseudovirus. Figure 5B The results showed that compared with the existing kozak-eGFP-2A-P1 design strategy, the pseudovirus titer of kozak-P1 was increased to varying degrees in different cell lines. The pseudovirus titer in RD, HEK 293T, Hep2, Vero, and Huh-7 cells increased by 4.1, 3.1, 5.0, 2.9, and 4.8 times, respectively.

[0182] The optimized capsid protein expression plasmid of the present invention was combined with the above-mentioned different cell lines, and the results were as follows: Figure 5C The results showed that compared with kozak-eGFP-2A-P1 and RD cells commonly used in the literature, the pseudovirus titer in the design strategy of the present invention (kozak-P1) and HEK 293T cell infection experiment increased by 27.8 times; compared with kozak-eGFP-2A-P1 and Hep2 cells in the live virus neutralization experiment, the pseudovirus titer in the design strategy of the present invention (kozak-P1) and HEK 293T cell infection experiment increased by 29.5 times.

[0183] In summary, the present invention found that it is best to use E2, eGFP and RFP fluorescent reporter genes for polio type 1, type 2 and type 3, respectively, and the optimal transfection ratio of capsid protein expression plasmid, replicon plasmid and T7 RNA polymerase expression plasmid is 3:3:1. In addition, by optimizing the P1 expression plasmid and using the sensitive cell HEK 293T system, the present invention successfully prepared high-titer polio fluorescent pseudoviruses; they were named Sabin1-E2 pseudovirus, Sabin2-eGFP pseudovirus and Sabin3-RFP pseudovirus, respectively.

[0184] Example 2: Establishment of a high-throughput detection method for simultaneous detection of three types of polio neutralizing antibodies

[0185] Neutralizing antibodies were detected using the prepared polio fluorescent pseudovirus, and parameters such as detection time, cell addition amount, and pseudovirus addition amount of the neutralization test were optimized. The detection sensitivity, specificity, and repeatability were verified, and the correlation between the neutralization test results of pseudovirus and live virus was analyzed. A high-throughput detection method for polio neutralizing antibodies with higher throughput, sample saving, economy, and convenience was established.

[0186] 1. Optimize detection time

[0187] Taking Sabin2-eGFP fluorescent pseudovirus as an example, the number of fluorescent spots was detected at different time points (6h, 12h, 16h, 20h, 24h, 36h and 72h) after the pseudovirus infected HEK 293T cells. The results are shown in Figure 6 The results showed that the number of fluorescent spots reached the maximum value 12 hours after infection and then tended to be stable, so the best detection time was selected as 12 hours after infection.

[0188] 2. Optimize the optimal amount of cells added

[0189] Taking the rat serum immunized with Sabin2-eGFP fluorescent pseudovirus and Sabin poliomyelitis inactivated vaccine monovalent stock solution (vaccine name: Sabin poliomyelitis inactivated vaccine monovalent stock solution, manufacturer: Institute of Medical Biology, Chinese Academy of Medical Sciences, immunization dose per animal: Type 2: 32DU) as an example, the serum sample was inactivated and diluted 3 times. 100 μl of the diluted sample and 50 μl of pseudovirus were added to each well of a 96-well plate. After incubation at 37°C for 1 hour, HEK 293T cells (5×10 3 , 1×10 4 , 2×10 4 , 4×10 4 , 8×10 4 and 1.6×10 5 After culturing at 37°C and 5% CO2 for 12 h, the number of fluorescent spots was detected and the ED was calculated using the Reed-Muench method or nonlinear regression.50 , E.D. 50 The dilution factor represents the sample that produces 50% inhibition.

[0190] The results are as follows Figure 7 The results showed that the linear correlation coefficient R 2 >0.98, indicating that the linear curve fits well. 50 Considering that the amount of cells added is 4×10 4 The curve fit is best when the number of holes is 1 / hole (R 2 =0.9942), the amount of cells added was set at 4×10 4 Pieces / hole.

[0191] 3. Optimize the amount of pseudovirus added

[0192] Taking the rat serum immunized with Sabin2-eGFP fluorescent pseudovirus and Sabin poliomyelitis inactivated vaccine monovalent stock solution (vaccine name: Sabin poliomyelitis inactivated vaccine monovalent stock solution, manufacturer: Institute of Medical Biology, Chinese Academy of Medical Sciences, immunization dose per animal: Type 2: 32DU) as an example, the serum sample was inactivated and diluted 3 times, and 100 μl of the diluted sample and 50 μl of pseudovirus were added to each well of a 96-well plate (the pseudovirus addition amounts were 40000, 12000, 4000, 1500, 500 and 150 TCID 50 After incubation at 37°C for 1 h, 4 × 10 4 HEK 293T cells were used, and the number of fluorescent spots was detected after 12 h. The ED was calculated using the Reed-Muench method or nonlinear regression. 50 , E.D. 50 represents the dilution factor of the sample when 50% inhibition occurs. Figure 8 The results show that the linear correlation coefficient R 2 >0.91, indicating that the linear curve fit is good. In addition, under different amounts of pseudovirus, ED 50 The values ​​are relatively close. Since the amount of pseudovirus added is 1500TCID 50 The curve fit is best when 2 =0.9947), and the amount of pseudovirus used was small, so the amount of pseudovirus added was determined to be 1500 TCID 50 / hole.

[0193] 4. Validation of the sensitivity of the fluorescent pseudovirus neutralization assay

[0194] The sensitivity of the established neutralizing antibody detection method was tested using Sabin1-E2 pseudovirus, Sabin2-eGFP pseudovirus, Sabin3-RFP pseudovirus, 20 rat negative sera, 25 mouse negative sera, and 11 New Zealand rabbit negative sera. The neutralization test operation was similar to the above. 100 μl of serially diluted serum samples were added to a 96-well plate, and 50 μl of fluorescent pseudovirus (1500 TCID 50 ), incubated at 37°C for 1 h, and then HEK 293T cells (4×10 4 After culturing at 37°C and 5% CO2 for 12 h, the number of fluorescent spots was detected and the ED was calculated using the Reed-Muench method or nonlinear regression. 50 , LOD (lower limit of detection) = ED 50 The lower limit of detection of neutralizing antibodies of different types of polio is as follows: Fig. 9 As shown, the cut off value is finally set to 15.

[0195] 5. Specificity Verification of Fluorescent Pseudovirus Neutralization Detection Method

[0196] The neutralization test was performed similarly to the above. 100 μl of serially diluted serum samples were added to a 96-well plate, and 50 μl of pseudovirus (a mixture of three fluorescent pseudoviruses at a ratio of 1:1:1, 1500 TCID 50 ), incubated at 37°C for 1 h, and then HEK 293T cells (4×10 4 After culturing at 37°C and 5% CO2 for 12 h, the number of fluorescent spots was detected and the ED was calculated using the Reed-Muench method or nonlinear regression. 50 .

[0197] (1) Verification of specificity between poliovirus types

[0198] The neutralizing titers of rat sera from 15 monovalent stock solutions of Sabin strain inactivated poliomyelitis vaccine (vaccine name: Sabin strain inactivated poliomyelitis vaccine monovalent stock solution, manufacturer: Institute of Medical Biology, Chinese Academy of Medical Sciences, immunization dose for each animal: type 1: 30DU; type 2: 32DU; type 3: 45DU) against three types of polio viruses were tested.

[0199] The results are as follows Fig. 10A As shown. The results showed that the Sabin1 type vaccine monovalent stock solution post-immunization serum can only neutralize Sabin1-E2 pseudovirus, and the Sabin2 type vaccine monovalent stock solution post-immunization serum can only neutralize Sabin2-eGFP pseudovirus. Similarly, the Sabin3 type vaccine monovalent stock solution post-immunization serum can only neutralize Sabin3-RFP pseudovirus. The results show that this method has good inter-type specificity.

[0200] (2) Specificity verification between different viruses

[0201] The neutralization titers of enterovirus antibody positive sera (mice) against Coxsackie virus group A type 6 (CV-A6), Coxsackie virus group A type 10 (CV-A10), Coxsackie virusgroup A type 16 (CV-A16) and enterovirus type 71 (EV71), SARS-CoV-2 antibody positive sera (guinea pigs) and H3N2 antibody positive sera (guinea pigs) against three types of poliovirus were tested respectively. Polio positive antibody serum (rabbit) was used as a positive control.

[0202] The results are as follows Fig. 10B The results showed that only polio antibody positive could neutralize three types of polio pseudoviruses, while other sera could not neutralize polio pseudoviruses, indicating that this method has good specificity among different viruses.

[0203] 6. Repeatability verification of the fluorescent pseudovirus neutralization detection method

[0204] The established fluorescent pseudovirus neutralization detection method was used to test the potency of the rabbit serum after immunization with a trivalent semi-finished Sabin strain polio vaccine (manufacturer: Institute of Medical Biology, Chinese Academy of Medical Sciences, immunization dose for each animal: type 1: 30DU; type 2: 32DU; type 3: 45DU). The test was repeated three times independently, with 6 replicates each time, and the CV (average coefficient of variation) within and between experiments was calculated.

[0205] The results are as follows Fig.11 The results showed that the intra-assay CVs of Sabin type 1, Sabin type 2, and Sabin type 3 neutralizing antibody detection were 12.52%, 12.08%, and 11.18%, respectively, and the inter-assay CVs were 13.39%, 14.65%, and 11.87%, respectively, indicating that the method had good repeatability.

[0206] 7. Correlation between the results of fluorescent pseudovirus neutralization test and live virus neutralization test

[0207] The three types of neutralizing antibodies were detected in 48 sera of Sabin strain poliomyelitis inactivated vaccine trivalent semi-finished products (manufacturer: Institute of Medical Biology, Chinese Academy of Medical Sciences, immunization dose per animal: type 1 30DU; type 2: 32DU; type 3: 45DU) using the established polio fluorescent pseudovirus neutralization detection method and live polio virus neutralization detection method (cytopathic method). Among the 42 serum samples, 15 were rabbit-derived sera and 27 were rat-derived sera. The neutralization test results of live virus and fluorescent pseudovirus were compared.

[0208] The results are as follows Fig.12 The results showed that for Sabin type 1, Sabin type 2, and Sabin type 3 neutralizing antibodies, the correlation between the fluorescent pseudovirus neutralization test results and the live virus test results were 0.8805, 0.9648, and 0.9703, respectively, suggesting that this method has a strong correlation with the live virus neutralization test results.

[0209] In summary, the method for simultaneous detection of three types of neutralizing antibodies of polio virus established by the present invention has good sensitivity, specificity, and repeatability, and has a good correlation with the detection results of live virus. Compared with the existing live polio virus neutralization detection method and the pseudovirus neutralization detection method based on chemiluminescent reporter gene, this method has many advantages such as high detection throughput, sample saving, low cost, convenient operation, and automated detection, and provides technical means for large-scale population polio virus neutralization antibody detection, vaccine research and development evaluation, etc.

[0210] References

[0211] 1.World Health Organization.Biologicals,U.,Manual of laboratorymethods for potency testing of vaccines used in the WHO Expanded Program onImmunization.1995,World Health Organization:Geneva.

[0212] 2. Liu, S., et al., A safe and reliable neutralization assay based on pseudovirus to measure neutralizing antibody titer against poliovirus. JMedVirol, 2017.89(12):p.2075-2083.

[0213] 3.Arita,M.,et al.,Development of a poliovirus neutralization testwith poliovirus pseudovirus for measurement of neutralizing antibody titer inhuman serum.Clin Vaccine Immunol,2011.18(11):p.1889-94.

[0214] 4.Jiang,Z.,et al.,A simple and safe antibody neutralization assaybased on polio pseudoviruses.Hum Vaccin Immunother,2019.15(2):p.349-357.

[0215] 5.Arita,M.and M.Iwai-Itamochi,Evaluation of antigenic differencesbetween wild and Sabin vaccine strains of poliovirus using the pseudovirusneutralization test.Sci Rep,2019.9(1):p.11970.

[0216] 6.Liu,S.,et al.,Comparison of the neutralizing activities ofantibodies in clinical sera against both Sabin and wild-type poliopseudoviruses.J Virol Methods,2022.300:p.114376.

[0217] 7.Arita,M.and M.Iwai-Itamochi,High-throughput analysis of anti-poliovirus neutralization antibody titre in human serum by the pseudovirusneutralization test.Sci Rep,2022.12(1):p.16074.

[0218] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A plasmid combination, comprising a poliovirus capsid protein expression plasmid, a poliovirus replicon plasmid and an RNA polymerase expression plasmid, wherein: The poliovirus capsid protein expression plasmid is a eukaryotic expression plasmid of the poliovirus P1 gene (e.g., the P1 gene of type 1, type 2, or type 3 poliovirus); The poliovirus replicon plasmid includes, in sequence: a promoter, a 5'UTR, a fluorescent reporter gene, a 2A peptide site, a P2 gene of a poliovirus (e.g., a P2 gene of a type 1, type 2, or type 3 poliovirus), a P3 gene of a poliovirus (e.g., a P3 gene of a type 1, type 2, or type 3 poliovirus), a 3'UTR, and Poly A, but does not include a poliovirus P1 gene.

2. The plasmid combination according to claim 1, characterized in that Any one or more of the following (1) to (15): (1) The eukaryotic expression plasmid is independently pcDNA3.1, pcDNA3.2, pcDNA3.3 or pcDNA3.4; (2) the promoter is a T7 promoter and the RNA polymerase is a T7 RNA polymerase, The promoter is a T3 promoter and the RNA polymerase is a T3 polymerase, or, The promoter is an SP6 promoter and the RNA polymerase is an SP6 polymerase; (3) The fluorescent reporter gene is one or more selected from eGFP, ZsGreen, YPet, RFP, mOrange, dTomato, mCherry, mKate2 and E2; (4) the 2A peptide is one or more selected from 2Apro, P2A, T2A, E2A and F2A; (5) the 5'UTR is derived from the poliovirus genome; (6) the 3'UTR is derived from the poliovirus genome; (7) the 5'UTR comprises the sequence shown in SEQ ID NO:4; (8) the 3'UTR comprises the sequence shown in SEQ ID NO:5; (9) The promoter comprises the sequence shown in SEQ ID NO:6; (10) The Poly A comprises the sequence shown in SEQ ID NO:7; (11) the 2A peptide site comprises the sequence shown in SEQ ID NO: 8; (12) The poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3; (13) The type 1 poliovirus is one or more selected from the group consisting of the type 1 Mahoney strain and Genbank Nos. AY184219 (Sabin 1), AF405671, KC880365 and AY518727, The type 2 poliovirus is one or more selected from the group consisting of type 2 MEF-1 strain and Genbank Nos. AY184220 (Sabin2), OP410365, MZ150566 and MG212459. The type 3 poliovirus is one or more selected from the group consisting of type 3 Saukett strain and Genbank Nos. AY184221 (Sabin3), OP137321, MG212494 and MG212445; (14) The ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1; (15) The poliovirus capsid protein expression plasmid does not contain a sequence encoding eGFP-2A.

3. A plasmid product, comprising a first plasmid combination, a second plasmid combination and a third plasmid combination, wherein the first plasmid combination, the second plasmid combination and the third plasmid combination are the plasmid combinations according to any one of claims 1 to 2, wherein: The first plasmid combination includes a type 1, type 2 or type 3 poliovirus capsid protein expression plasmid, a type 1 poliovirus replicon plasmid and an RNA polymerase expression plasmid; The second plasmid combination includes a type 1, type 2 or type 3 poliovirus capsid protein expression plasmid, a type 2 poliovirus replicon plasmid and an RNA polymerase expression plasmid; The third plasmid combination includes a type 1, type 2 or type 3 poliovirus capsid protein expression plasmid, a type 3 poliovirus replicon plasmid and an RNA polymerase expression plasmid; The first plasmid combination, the second plasmid combination and the third plasmid combination are independent of each other or mixed together; The fluorescent reporter genes in the first plasmid combination, the second plasmid combination and the third plasmid combination are different from each other and can emit different fluorescence; Preferably, the type 1 poliovirus is one or more selected from the group consisting of type 1 Mahoney strain and Genbank Nos. AY184219 (Sabin 1), AF405671, KC880365 and AY518727. The type 2 poliovirus is one or more selected from the group consisting of type 2 MEF-1 strain and Genbank Nos. AY184220 (Sabin2), OP410365, MZ150566 and MG212459. The type 3 poliovirus is one or more selected from the group consisting of type 3 Saukett strain and Genbank Nos. AY184221 (Sabin3), OP137321, MG212494 and MG212445; Preferably, none of the poliovirus capsid protein expression plasmids in the three plasmid combinations contains the eGFP-2A sequence.

4. The plasmid product according to claim 3, wherein The fluorescent reporter gene is selected from eGFP, RFP and E2; Preferably, the fluorescent reporter genes in the first plasmid combination, the second plasmid combination and the third plasmid combination are E2, eGFP and RFP respectively; Preferably, E2 comprises the sequence shown in SEQ ID NO: 1; Preferably, eGFP comprises the sequence shown in SEQ ID NO: 2; Preferably, RFP comprises the sequence shown in SEQ ID NO:

3.

5. A method for preparing poliomyelitis pseudovirus, comprising the step of transfecting eukaryotic cells with the plasmid combination according to any one of claims 1 to 2 or the plasmid product according to any one of claims 3 to 4; Preferably, the eukaryotic cells are selected from one or more of HEK 293T, RD, Hep2, Vero and Huh-7 cells; Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:

1.

6. A polio pseudovirus prepared by the method of claim 5; Preferably, the polio pseudovirus is type 1-E2 polio pseudovirus, type 2-eGFP polio pseudovirus or type 3-RFP polio pseudovirus; Preferably, the polio pseudovirus is Sabin1-E2 pseudovirus, Sabin2-eGFP pseudovirus or Sabin3-RFP pseudovirus; Preferably, the polio pseudovirus is a type 1 Mahoney strain-E2 pseudovirus, a type 2 MEF-1 strain-eGFP pseudovirus or a type 3 Saukett strain-RFP pseudovirus.

7. A polio pseudovirus combination, comprising polio pseudovirus type 1, polio pseudovirus type 2 and polio pseudovirus type 3, which are obtained by transfecting eukaryotic cells with the first plasmid combination, the second plasmid combination and the third plasmid combination in the plasmid product of any one of claims 3 to 4, respectively. The type 1 polio pseudovirus, type 2 polio pseudovirus and type 3 polio pseudovirus are independent of each other or mixed together; Preferably, the eukaryotic cells are selected from one or more of HEK 293T, RD, Hep2, Vero and Huh-7 cells; Preferably, the ratio of the poliovirus capsid protein expression plasmid, the poliovirus replicon plasmid and the RNA polymerase expression plasmid in the plasmid combination is (1-3):(1-3):(1-3), for example, 3:3:1, 1:3:1, 1:2:1 or 1:1:1; Preferably, the polio pseudovirus combination includes type 1-E2 pseudovirus, type 2-eGFP pseudovirus and type 3-RFP pseudovirus; Preferably, the polio pseudovirus combination includes Sabin1-E2 pseudovirus, Sabin2-eGFP pseudovirus and Sabin3-RFP pseudovirus; Preferably, the polio pseudovirus combination includes type 1 Mahoney strain-E2 pseudovirus, type 2 MEF-1 strain-eGFP pseudovirus and type 3 Saukett strain-RFP pseudovirus.

8. A method for detecting poliovirus neutralizing antibodies for non-therapeutic and non-diagnostic purposes, comprising the following steps: (1) contacting or incubating an appropriate amount of the polio pseudovirus described in claim 6 or the polio pseudovirus combination described in claim 7 with a sample to be tested; (2) adding sensitive cells and continuing to culture for a period of time, and detecting the number of fluorescent spots; and (3) Calculate ED based on the number of fluorescent spots 50 ; Preferably, the method further comprises the following steps: (4) The obtained ED 50 Compare with the cut off value.

9. The method according to claim 8, characterized in that Any one or more of the following items ① to ⑨: ① The sample to be tested in step (1) is a serum sample; ② The amount of polio pseudovirus in step (1) is 1000 TCID 50 Up to 3500TCID 50 , preferably 1000TCID 50 Up to 3000TCID 50 , 1000TCID 50 Up to 2500TCID 50 、1000TCID 50 Up to 2000TCID 50 or 1500TCID 50 ; ③ The incubation in step (1) is at 37° C. for 0.5-3 h, for example, 1-2 h; ④ The sensitive cells in step (2) are HEK 293T cells; ⑤In step (2), the amount of sensitive cells used is 1500 TCID 50 Polio pseudovirus 5×10 3 Up to 8×10 4 cells / well; preferably 4×10 4 Up to 6×10 4 cells / well; ⑥ In step (2), the culture is carried out for 6-72 hours, preferably 12-24 hours; ⑦ In step (3), the Reed-Muench method or nonlinear regression is used to calculate ED 50 ; ⑧ In step (4), the cut-off value for pseudovirus neutralization detection is 15; ⑨The method is for pharmaceutical purposes.

10. Use of the polio pseudovirus according to claim 6 or the polio pseudovirus combination according to claim 7 in the preparation of a drug for detecting polio virus neutralizing antibodies, evaluating the efficacy of polio vaccination, diagnosing polio virus infection or monitoring polio virus transmission.

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