Construction and application of recombinant RSV infectious clone

Abstract

The invention relates to construction and application of a recombinant RSV infectious clone, Long-BAC is taken as a basic skeleton, an F gene of a Long strain is respectively replaced with an F gene of an RSV A subtype global preponderant epidemic genotype ON1 and an F gene of a B subtype BA9 genotype, and two attenuated recombinant virus strains are obtained through rescue by a reverse genetic technology. The technical method disclosed by the invention has the advantages of rapidness and accuracy, the recombinant virus containing the epidemic genotype F gene can be successfully constructed, and the recombinant virus provides a scientific and technical platform for researching the influence of F protein variation on virus transmission capacity, host adaptability and pathogenicity and for neutralizing and escaping evaluation of vaccines and antibody drugs.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to the construction and application of a recombinant RSV infectious clone. Background Art

[0002] Respiratory Syncytial Virus (RSV) is one of the most important pathogens causing acute lower respiratory tract infections in children under five years of age and immunocompromised individuals worldwide. Currently, RSV vaccines based on various technologies are under development. Three vaccines have received FDA approval for the prevention of RSV infection in people aged 60 years and older: Arexvy (RSVPreF3OA / GSK3844766A, developed by GSK), ABRYSVO (RSVpreF / PF-06928316, developed by Pfizer), and mRNA-1345 (RSVpreF, developed by Moderna). With the rapid advancement of passive immunization technology, the monoclonal antibody Nirsevimab has been approved in both the EU and the US for the prevention of first infection in newborns and infants during the RSV epidemic season. Nirsevimab is a recombinant human IgG1 kappa monoclonal antibody that binds to highly conserved epitopes on the F1 and F2 subunits of the RSV F protein, effectively preventing viral infection.

[0003] RSV (Respiratory Syndrome Virus) is a single-stranded, negative-sense RNA virus belonging to the Pneumoviridae family and the genus Orthopneumovirus. Its genome is approximately 15.2 kb long and encodes 11 proteins. Two major glycoproteins, the adhesion protein (G protein) and the fusion protein (F protein), are important viral antigenic proteins and the primary viral antigens that induce neutralizing antibodies. RSV has only one serotype, which is divided into two subtypes based on antigenicity: RSV A and RSV B. Due to the high genetic variability of the nucleotide sequence at the 3' end of the second hypervariable region (G-HVR2) of the G protein, RSV genotypes are generally classified internationally based on G-HVR2. RSV A is divided into 15 genotypes (GA1-GA7, SAA1, NA1-4, ON1-2, and CB-A), and RSV B into 28 genotypes (BA1-BA14, SAB1-SAB4, GB1-GB4, BAc, CB1, BA, THB, and URU1-2). The RSV Long strain is the prototype strain of RSV-A type and plays an important role in the research and prevention of RSV. At present, the ON1 and BA9 genotypes are the main dominant genotypes in the world.

[0004] The RSV F protein is a highly conserved, N-glycosylated, type I transmembrane protein that mediates fusion between the virus and host cell membranes, promoting further viral transmission and inducing the production of highly effective protective antibodies. It plays a crucial role in viral infection and immune response and is currently the primary target protein for the development of vaccines, monoclonal antibodies, and small molecule drugs. Therefore, understanding the genetic variation and antigenic changes in the F protein sequence among contemporary circulating RSV strains is crucial.

[0005] Since RSV virus isolation requires fresh specimens and bedside inoculation, it is difficult to obtain isolates. However, most RSV vaccine research and development are based on laboratory strains, which cannot reflect the impact of the variation of prevalent strains on vaccine and antibody efficacy. In addition, existing systems lack a robust platform that can be used for neutralizing antibody testing. In response to these technical difficulties, the present invention proposes an attenuated RSV recombinant virus based on reverse genetic platform technology to mutate the F protein of prevalent strains ON1 and BA9, and can be applied to the preparation, screening and / or evaluation of neutralizing antibodies such as potential vaccines and Nirsevimab. Summary of the Invention

[0006] To overcome the above-mentioned drawbacks, the present invention provides a recombinant RSV strain based on a reverse genetics system and a method for its construction. Specifically targeting the F protein variants of the currently prevalent RSV strains ON1 and BA9 genotypes, a recombinant virus strain was constructed, which is further used as an application platform for neutralizing antibody evaluation and drug development. The details are as follows:

[0007] In a first aspect, the present invention provides a recombinant RSV strain, wherein the recombinant RSV strain comprises an RSV genome or antigenome derived from an RSV Long strain, wherein the F protein of the Long strain is replaced with an F protein of another RSV genotype strain.

[0008] Preferably, the other RSV genotype strains include any genotype of RSV A subtype or RSV B subtype, such as any one of GA1-GA7, SAA1, NA1-4, ON1-2 and CB-A genotypes in RSV A subtype, or any one of BA1-BA14, SAB1-SAB4, GB1-GB4, BAc, CB1, BA, THB and URU1-2 genotypes in RSV B subtype. More preferably, the other RSV genotype strains include RSV ON1 genotype or BA9 genotype strain.

[0009] More preferably, the amino acid sequence of the RSV Long strain F protein is shown in GenBank: AAX23994.1. The amino acid sequence of the RSV ON1 genotype strain F protein is shown in SEQ ID NO: 8. The amino acid sequence of the RSV BA9 genotype strain F protein is shown in SEQ ID NO: 9.

[0010] More preferably, the gene sequence encoding the RSV Long strain F protein is shown in SEQ ID NO: 10. The gene sequence encoding the RSV ON1 genotype strain F protein is shown in SEQ ID NO: 11. The gene sequence encoding the RSV BA9 genotype strain F protein is shown in SEQ ID NO: 12.

[0011] Preferably, the recombinant RSV strain comprises codon-optimized N, P, L and M2-1 genes, wherein:

[0012] The N gene includes nucleotide sequence 923-2104 shown in SEQ ID NO: 1;

[0013] The P gene includes nucleotide sequence 923-1654 shown in SEQ ID NO: 2;

[0014] The L gene includes nucleotide sequence 923-7426 shown in SEQ ID NO: 3;

[0015] The M2-1 gene includes positions 923-1513 of the nucleotide sequence shown in SEQ ID NO:4.

[0016] In a second aspect, the present invention provides a biomaterial, comprising:

[0017] (1) A vector comprising the gene sequence of the above-mentioned recombinant RSV strain;

[0018] (2) A cell, characterized in that the cell comprises the above-mentioned recombinant RSV strain or the vector in (1).

[0019] The vector described herein refers to a vector that can carry exogenous DNA, mRNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids (bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), P1-derived artificial chromosomes (PAC), F plasmids, R plasmids, Col plasmids, Ti plasmids, expression plasmids, shuttle plasmids, cloning plasmids, etc.), bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0020] Preferably, the vector comprises a BAC vector.

[0021] Preferably, the vector further comprises regulatory elements, such as a promoter for initiating transcription of the above nucleotide sequence, a terminator for terminating transcription of the above nucleotide sequence, an enhancer sequence or other regulatory sequences that are beneficial to splicing or sequence stability.

[0022] In one embodiment, the regulatory elements include a T7 promoter, a hammerhead ribozyme sequence, a hepatitis D ribozyme sequence, a T7 terminator, and a fragment of a bovine growth hormone (BGH) polyadenylation signal.

[0023] In one embodiment, the vector comprises, from 5' to 3' end, a T7 promoter, a hammerhead ribozyme sequence, all or part of the sequence of the relevant gene of the RSV strain or recombinant RSV strain, hepatitis D ribozyme and a T7 terminator sequence.

[0024] The cells described herein include host cells (also referred to as recipient cells) and are understood to refer not only to the specific recipient cells, but also to the progeny of such cells. Due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but are still included in the scope of host cells. Suitable cells are known in the art, wherein: the plant cells may be plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but are not limited thereto; the animal cells may be mammalian cells (such as Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), golden hamster kidney cells (BSR cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, human laryngeal epidermoid carcinoma cells (HEp-2 cells), fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (such as chicken or duck cells), amphibian cells (such as African clawed frog (Xenopus laevis) cells or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells or Sf-9 cells), etc., but are not limited thereto.

[0025] Preferably, the host cell may also be a microorganism, and the microorganism described herein may be a bacterium, a fungus, an actinomycete, a protozoa, an algae, or a virus. The bacterium may be from the genus Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but is not limited thereto. For example, the bacterium may be Escherichia coli, Bacillus subtilis, or Bacillus pumilus.

[0026] In one embodiment, the cells comprise BSR T7 / 9 or HEp-2 cells.

[0027] In a third aspect, the present invention provides a method for constructing the above-mentioned recombinant RSV strain, wherein the method comprises replacing the F gene sequence in the RSV Long strain with the F gene sequence of another RSV genotype strain.

[0028] Preferably, the method comprises replacing the F gene sequence in the RSV Long strain with the F gene sequence of the RSV ON1 or BA9 genotype strain.

[0029] Preferably, the method comprises:

[0030] 1) constructing the above-mentioned vector containing the gene sequence of the recombinant RSV strain;

[0031] 2) constructing an auxiliary vector, wherein the auxiliary vector comprises the N, P, L or M2-1 gene;

[0032] 3) Virus rescue: Using a reverse genetics method, the vector of step 1) and the auxiliary vector of step 2) are co-transfected into cells, and the virus is harvested to obtain a recombinant RSV strain.

[0033] More preferably,

[0034] The step 1) includes

[0035] 1-1) constructing a vector comprising a Long strain genomic fragment;

[0036] 1-2) Construct an F gene sequence replacement vector to replace the F gene sequence of the RSV Long strain with the F gene sequence of other RSV genotype strains.

[0037] More preferably,

[0038] Preferably, in step 2),

[0039] The auxiliary vector includes the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 4.

[0040] The N gene includes nucleotide sequence 923-2104 shown in SEQ ID NO: 1;

[0041] The P gene includes nucleotide sequence 923-1654 shown in SEQ ID NO: 2;

[0042] The L gene includes nucleotide sequence 923-7426 shown in SEQ ID NO: 3;

[0043] The M2-1 gene includes positions 923-1513 of the nucleotide sequence shown in SEQ ID NO:4.

[0044] More preferably, the method comprises:

[0045] 1-1) constructing a vector comprising a T7 promoter, hammerhead ribozyme, the complete genome sequence of the Long strain, the hepatitis D ribozyme, and a T7 terminator sequence;

[0046] 1-2) Replacing the F gene sequence of RSV ON1 or BA9 strain with the F gene sequence of RSV Long strain to obtain a recombinant vector;

[0047] 2) constructing auxiliary vectors containing the codon-optimized N, P, L, and M2-1 genes respectively;

[0048] 3) Virus rescue: using reverse genetics, the vectors from steps 1-2) and the auxiliary vector from step 2) are co-transfected into cells, and the viruses are harvested to obtain recombinant RSV strains.

[0049] Further preferably, the virus rescue comprises co-transfecting the above-mentioned vector and auxiliary vector into cells, culturing, observing cytopathic conditions, collecting cells, freezing and thawing, centrifuging, collecting the supernatant and continuing to passage it into another cell.

[0050] In one embodiment, the virus rescue comprises co-transfecting the above-mentioned vector and the helper vector into BSR T7 / 9 cells; continuing to culture, observing the BSR T7 / 9 cells for obvious fusion lesions, collecting the cells, freezing and thawing, centrifuging, collecting the supernatant, and then continuing to passage it into HEp-2 cells.

[0051] In a fourth aspect, the present invention provides a reverse genetics system for RSV strains, wherein the reverse genetics system comprises the vector in the above-mentioned biological material and an auxiliary vector, wherein the auxiliary vector comprises the N, P, L or M2-1 gene,

[0052] More preferably,

[0053] The auxiliary vector includes the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 4.

[0054] The N gene includes nucleotide sequence 923-2104 shown in SEQ ID NO: 1;

[0055] The P gene includes nucleotide sequence 923-1654 shown in SEQ ID NO: 2;

[0056] The L gene includes nucleotide sequence 923-7426 shown in SEQ ID NO: 3;

[0057] The M2-1 gene includes positions 923-1513 of the nucleotide sequence shown in SEQ ID NO:4.

[0058] In a fifth aspect, the present invention provides applications of the aforementioned recombinant RSV strain, the aforementioned biological material, the recombinant RSV strain obtained by the aforementioned construction method, or the aforementioned reverse genetics system, wherein the applications include any of the following:

[0059] (1) Application in the preparation or screening of products for detecting, preventing and / or treating diseases caused by RSV infection;

[0060] (2) Application in the preparation or screening of products for detecting and inducing immune responses to RSV antigens.

[0061] Preferably, the products include detection reagents, drugs, etc., and the drugs may be vaccines.

[0062] Preferably, the RSV infection includes infection caused by the ON1 genotype of RSV A subtype or the BA9 genotype of RSV B subtype.

[0063] In a sixth aspect, the present invention provides a method for evaluating RSV neutralizing antibodies, which comprises incubating the above-mentioned recombinant RSV strain or the recombinant RSV strain obtained by the above-mentioned construction method with the antibody to be tested and then conducting a neutralization assay.

[0064] Preferably, the method is an in vitro detection method, not a disease diagnostic method. For example, the method can be used for scientific research purposes, preclinical drug evaluation methods, including but not limited to evaluation methods for viral virulence, vaccine-induced antibody effects, antibody drug effects, etc.

[0065] In a seventh aspect, the present invention provides a reagent comprising the aforementioned recombinant RSV strain, the aforementioned biological material, or the aforementioned reverse genetics system.

[0066] Preferably, the reagent comprises a buffer.

[0067] Preferably, the agent can be used as a drug.

[0068] Preferably, the drug can be therapeutic or prophylactic.

[0069] More preferably, the drug is a vaccine.

[0070] Further preferably, the vaccine further comprises an adjuvant.

[0071] More preferably, the adjuvant can be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein can be well known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factors IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant can be a pharmaceutically acceptable adjuvant.

[0072] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0073] Beneficial effects of the present invention:

[0074] 1. The present invention utilizes reverse genetics technology to build RSV Long strain RSV reverse genetics system with low copy bacterial artificial chromosome (Bacterial artificial chromosome, BAC) vector as skeleton. Use BAC vector to effectively reduce the mismatch rate of RSV genome of up to 15kb in Escherichia coli replication, while BAC vector can remain on a high level in transfection and expression efficiency, ensure the accuracy of the virus full genome, after genome transfection cells, can express and virus assembly rapidly, for later stage transformation provides stable cDNA template.

[0075] 2. RSV is a virus that has been difficult to effectively prevent and control for a long time, and its genotype variation has an important impact on the research and development of vaccines and antibody therapies. At present, there are no relevant reports on the stable expression and functional verification of ON1 and BA9 F genes. The present invention uses reverse genetic technology, based on Long-BAC as the basic framework, to replace the F gene of the Long strain with the F gene of the global dominant prevalent genotype RSV A subtype ON1 genotype, and the F gene of the RSV B subtype BA9 genotype, respectively, to rescue and obtain two recombinant virus strains, in order to explore the effects of its F protein variation on the virus's transmissibility, host adaptability and pathogenicity, and to provide scientific data support for the neutralization escape evaluation of vaccines and antibody drugs currently under development.

[0076] 3. In the preferred embodiment, codon optimization improves the efficiency and stability of auxiliary protein expression and supports the efficient operation of the reverse genetics system. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is a strategy for constructing Long-BAC full-length genomic cDNA and recombinant virus strains.

[0078] Figure 2 This is the result of indirect immunofluorescence identification of the rescued recombinant virus. Figures A and B correspond to the cytopathic effects of HEp-2 cells infected with Group 2 and Group 3, respectively, and Figure C corresponds to normal HEp-2 cells not inoculated with the virus.

[0079] Figure 3 Growth curve of the recombinant virus.

[0080] Figure 4 is the neutralizing antibody inhibition curve. DETAILED DESCRIPTION

[0081] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0082] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0083] 1. Main instruments

[0084] Automatic cell counter (Invitrogen, USA), inverted microscope (Olympus, Japan), standard CO2 incubator (Thermo Fisher Scientific, USA), water bath (model: OLS200) (Grant, Finland), high-speed refrigerated centrifuge (model: Primo) (Heraeus, Germany), 45-liter liquid nitrogen tank (model: XC-33 / 22) (MVE, USA), electronic balance (model: AS180) (Mettler, Japan).

[0085] 2. Strains, plasmids and main reagents

[0086] RSV Long strain (Long-WT) (ATCC VR26, preserved in 25% sucrose, its gene sequence is available in GenBank: AY911262.1) is maintained in our laboratory; BAC plasmid (plasmid maintained in our laboratory); pcDNA3.1(+) plasmid is maintained in our laboratory; viral RNA extraction kit and endotoxin-free plasmid extraction kit (QIAGEN, Germany); fetal bovine serum, DMEM, 2× DMEM, 0.25% trypsin digestion solution containing EDTA, Opti-MEM, and Lipofectamine 3000 (Hyclone, USA); ampicillin sodium and Tween-20 (Amresco, USA); Nirsevimab monoclonal antibody is maintained in our laboratory; FITC-labeled anti-human IgG was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0087] The amino acid sequence of the RSV Long strain F protein involved in the examples is shown in GenBank: AAX23994.1, the amino acid sequence of the RSV ON1 strain F protein is shown in SEQ ID NO: 8, the amino acid sequence of the RSV BA9 strain F protein is shown in SEQ ID NO: 9, the RSV Long strain F gene sequence is shown in SEQ ID NO: 10, and the RSV ON1 strain F gene sequence is shown in SEQ ID NO: 11. The encoding RSV BA9 strain F gene sequence is shown in SEQ ID NO: 12.

[0088] Example 1 Construction and rescue of recombinant RSV strains

[0089] 1RSV Long-BAC Construction Strategy

[0090] An infectious clone containing the full-length cDNA sequence of the RSV Long strain was constructed using a low-copy bacterial artificial chromosome (BAC) as a vector backbone. The T7 promoter, hammerhead ribozyme, the complete genome sequence of the Long prototype strain, hepatitis D ribozyme, and T7 terminator sequence were inserted from the 5′ to 3′ direction at the restriction sites BamHI and HindIII on the BAC plasmid to generate recombinant plasmid 1. Recombinant plasmid 1 was synthesized by Nanjing GenScript and named Long-BAC plasmid after sequencing verification.

[0091] 2 Construction strategy of recombinant plasmids Long-BJ1903-AF and Long-SY2103-BF

[0092] Based on the Long-BAC plasmid, the F gene sequence of the Long prototype strain was replaced by the F gene sequence of the ON1 and BA9 genotypes, respectively, to obtain recombinant plasmids 2 and 3. Recombinant plasmids 2 and 3 were synthesized by Nanjing GenScript and named recombinant plasmids Long-BJ1903-AF and Long-SY2103-BF after sequencing verification, indicating that the recombinant plasmids in which the F gene of the Long prototype strain was replaced by the F gene of the ON1 and BA9 genotypes, respectively.

[0093] The specific construction strategies of Long-BAC and recombinant plasmids are as follows: Figure 1 shown.

[0094] 3. Long-BAC, Long-BJ1903-AF and Long-SY2103-BF virus rescue

[0095] 3.1 Preparation of bacterial solution

[0096] Taking the Long-BAC plasmid as an example, the full-length recombinant plasmid was transformed into DH10B competent cells. To a 1.5mL Eppendorf (EP) tube, 50μL of DH10B competent medium was added, followed by 1μL of plasmid. Mix gently and incubate on ice for 30 minutes. The EP tube was heat-shocked at 42°C for 90 seconds, then quickly incubated on ice for 2 minutes. 500μL of resistance-free LB liquid medium was added and the cells were incubated at 37°C with shaking at 220 rpm for 1 hour. 50μL of the transformation solution was evenly spread onto a solid LB plate containing chloramphenicol and grown in a 37°C incubator for 16-20 hours. Five uniformly sized single colonies were selected and inoculated into 5mL of LB liquid medium containing the appropriate chloramphenicol resistance. The cells were cultured for 12-16 hours and then sequenced. The correct plasmid sequence was verified by bacterial culture sequencing. Verified strains were stored in 25% glycerol for expansion in the next experiment.

[0097] 3.2 Plasmid extraction

[0098] The positive bacterial solution containing the full-length recombinant plasmid 1 was inoculated into LB liquid medium with appropriate resistance at a dilution of 1:100. The cells were cultured at 37°C in a shaker at 220 rpm for 16-20 h. The plasmid was then extracted according to the instructions of the QIAGEN EndoFree Plasmid Maxi Kit (Cat. No. 12362). The specific steps are as follows:

[0099] Note before use: Add RNase A solution to buffer P1, mix and store at 2-8°C, add RNase A solution to buffer P1 at a ratio of 1:1000. Reagents (optional); pre-cool buffer P3 at 4°C; check buffer P2 for SDS precipitation; prepare isopropanol in advance.

[0100] (1) 250 mL of overnight Long-BAC culture was centrifuged in batches at 12,000 rpm for 15 min at 4°C to collect the precipitate.

[0101] (2) Add 10 mL of buffer P1 to the 50 mL centrifuge tube containing the collected bacterial precipitate;

[0102] (3) Add 10 mL of buffer P2 to a 50 mL centrifuge tube and gently invert the tube 6-8 times to mix thoroughly. After the bacteria are fully lysed, place the tube at room temperature (15-25°C) for 5 minutes. The solution will become clear and viscous. If LyseBlue reagent is used, the solution will turn blue.

[0103] (4) After standing at room temperature, screw the QIAfilter cartridge cap onto the QIAfilter cartridge outlet nozzle. Place the QIAfilter cartridge in the tube rack.

[0104] (5) Add 10 mL of ice-cold buffer P3 to the side of the centrifuge tube and immediately invert and rotate 4 to 6 times to mix thoroughly. When a white flocculent precipitate appears, mix thoroughly. If using LyseBlue reagent, mix the solution until it is completely colorless.

[0105] (6) Add the lysate to the QIAfilter cartridge. Incubate at room temperature for 10 min. Unscrew the cap from the QIAfilter cartridge outlet. Gently insert the plunger handle into the QIAfilter cartridge and collect the filtrate in a sterilized 50 mL collection tube.

[0106] (7) Add 2.5 mL of buffer ER to the filtered lysate, invert the tube 10 times to mix thoroughly, and incubate on ice for 30 min.

[0107] (8) Column equilibration: Equilibrate the QIAGEN-tip500 column with 10 mL of buffer QBT and drain the column by gravity flow;

[0108] (9) Add the liquid from step (7) to the QIAGEN-tip and allow it to enter the tip;

[0109] (10) Add 30 mL of buffer QC and wash the QIAGEN-tip (repeat twice);

[0110] (11) Elute the DNA with 15 mL of buffer QN into a 30 mL endotoxin-free or pyrogen-free tube;

[0111] (12) Add 10.5 mL of isopropanol to the filtrate from step (11) and mix. Centrifuge at 13,000 rpm for 30 min at 4°C and carefully pour off the supernatant.

[0112] (13) Add 5 mL of 70% ethanol to wash the DNA pellet and centrifuge at 13,000 rpm for 10 min at room temperature. Carefully aspirate the supernatant without disrupting the pellet.

[0113] (14) Air-dry the pellet for 5–10 min to completely dry the ethanol, and dissolve the DNA in an appropriate volume of endotoxin-free buffer TE to obtain a plasmid solution. After measuring the concentration and purity of the solution, aliquot and store at −40°C until use.

[0114] Long-BJ1903-AF and Long-SY2103-BF plasmids were obtained using the same method.

[0115] 3.3 Virus rescue

[0116] The N, P, L and M2-1 gene sequences of the Long prototype strain (GenBank No.: AY911262) were codon-optimized and connected to the pcDNA3.1(+) vector to construct four helper plasmids pcDNA3.1-N, pcDNA3.1-P, pcDNA3.1-M2-1 and pcDNA3.1-L. The optimized sequences are shown in SEQ ID NOs: 1-4, respectively.

[0117] BSR T7 / 9 cells were trypsinized and plated in six-well cell culture plates. When the cell confluence reached 80%, the following plasmids were co-transfected into BSR T7 / 9 cells according to the instructions of Lipofectamine 3000 transfection reagent:

[0118] Group 1: Long-BAC plasmid (2 μg), pcDNA3.1-N (1.5 μg), pcDNA3.1-P (0.75 μg), pcDNA3.1-M2-1 (0.25 μg), and pcDNA3.1-L (0.4 μg);

[0119] Group 2: Long-BJ1903-AF plasmid (2 μg), pcDNA3.1-P (0.75 μg), pcDNA3.1-N (1.5 μg), pcDNA3.1-M2-1 (0.25 μg), and pcDNA3.1-L (0.4 μg);

[0120] Group 3: Long-SY2103-BF plasmid (2 μg), pcDNA3.1-P (0.75 μg), pcDNA3.1-N (1.5 μg), pcDNA3.1-M2-1 (0.25 μg) and pcDNA3.1-L (0.4 μg).

[0121] The Long-BAC strain, the recombinant strain Long-BJ1903-AF and the recombinant strain Long-SY2103-BF were obtained.

[0122] At the same time, a plasmid transfection control group was set up (the positive control plasmid was pEGFP-C1, and the negative control plasmid was pPBR322-RSV-EGFP.

[0123] The cells were cultured in a 37°C, 5% CO2 incubator for 24-48 hours, and the expression of EGFP was observed under an inverted fluorescence microscope.

[0124] The specific steps of transfection are as follows:

[0125] (1) BSR T7 / 9 cells were cultured in six-well plates. When the cell confluence reached 80% in a monolayer, the medium in the wells was discarded, the cells were washed three times with PBS, and then replaced with DMEM maintenance medium (2% FBS, 1% double-antibody DMEM medium) for transfection.

[0126] (2) Take two sterile 1.5mL EP tubes, marked as A and B, and add 125μL of Opti-MEM to tube A. TM culture medium, then add 3.75 μL of 3000 reagent, mix thoroughly;

[0127] (3) Add 125 μL of Opti-MEM™ medium to tube B, add the plasmids of groups 1-3, and add 10 μL of P3000™ reagent to each of the groups. Gently stir the tube wall to mix and let it stand for 2-3 minutes.

[0128] (4) Slowly add the liquid in tube B to tube A, mix thoroughly, and incubate at 37°C for 15 min;

[0129] (5) Add the DNA-lipid complex in tube A along the wall of the six-well plate with cells, gently shake the six-well plate to mix the liquid in the wells, and place it in a 37°C, 5% CO2 incubator for incubation;

[0130] (6) Culture in a 37°C 5% CO2 incubator for 5–12 days, blindly passage for 3 generations, observe the appearance of CPE in the cells, freeze-thaw, and then centrifuge at 450 g for 5 min at 4°C to collect the supernatant;

[0131] (7) The supernatant was adsorbed on HEp-2 cells for 1 hour, and the supernatant was discarded after 1 hour. The cells were washed three times with PBS, and 2% maintenance solution (2% fetal bovine serum in DMEM medium) was added and cultured in a 37°C incubator for 4-5 days. The cell pathological changes were observed.

[0132] (8) Collect cells, freeze-thaw, centrifuge, collect the supernatant, and then continue to passage it into HEp-2 cells.

[0133] Example 2 Indirect immunofluorescence (IFA) identification of rescued viruses

[0134] HEp-2 cells were evenly plated into six-well plates. When the cell density reached 70% to 80%, the recombinant viruses of different groups in Example 1 were inoculated, adsorbed for 1 hour and discarded, and washed 3 times with PBS. After adding 2% maintenance liquid and culturing for 48 hours, the maintenance liquid was discarded and washed 3 times with PBS. The cells were fixed with pre-cooled fixative (methanol: acetone ratio of 1:1) for 15 minutes; blocked with 5% skim milk powder for 1 hour and washed 3 times; added palivizumab (1:5000 diluted in 5% skim milk) and incubated at 37°C for 1 hour, washed 3 times; added FITC-labeled goat anti-human IgG (1:400 diluted in 5% skim milk) and incubated at 37°C in the dark for 1 hour, washed 3 times, and observed under an inverted fluorescence microscope.

[0135] The results are as follows Figure 2 As shown, Figures A and B correspond to the cytopathic effects of HEp-2 cells infected in Group 2 and Group 3, respectively, and Figure C corresponds to normal HEp-2 cells that were not inoculated with the virus. The upper figure is a bright field observation figure, and the lower figure is a dark field observation figure.

[0136] It was observed that the recombinant rescued viruses (Group 2 and Group 3) caused specific syncytial lesions in the HEp-2 cell line and bound to specific antibodies.

[0137] Example 3 Virus Growth Curve Determination

[0138] (1) Cell preparation: One day before the experiment, HEp-2 cells were plated in a 6-well plate and counted at 1×10 5 Cells were cultured at a density of 100 cells / mL in a 37°C 5% CO2 incubator overnight until the cell density reached 80% to 90%.

[0139] (2) The Long-BAC strain (Group 1), the tenth generation of the two recombinant strains (Groups 2-3), and the Long-WT strain (original Long strain) were inoculated into HEp-2 cells prepared in advance at a multiplicity of infection (MOI) of 0.1;

[0140] (3) HEp-2 cells were infected with Long-BAC strain, the tenth generation recombinant strain, and Long-WT strain at different time points, and the cells were frozen and thawed once at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 90 h, centrifuged at 3000 rpm for 10 min, and cell debris was discarded. The supernatant was aspirated and stored at -80 °C.

[0141] (4) After all time points have been collected, total RNA is extracted. Quantification is performed using digital PCR (refer to the digital PCR method for specific steps) and a multi-step viral growth curve is plotted.

[0142] The specific steps of the digital PCR method are as follows:

[0143] Primer sequences are shown in Table 1:

[0144] Table 1 Primer and probe sequences for Real-time RT-PCR

[0145]

[0146] Note: F and R represent the forward primer and the reverse primer respectively, and probe represents the probe.

[0147] The reaction system (25 μL) was prepared as shown in Table 2 below:

[0148] Table 2 Preparation of RSV digital PCR reaction system

[0149]

[0150]

[0151] Note: μM is the concentration unit, 1 μM = 1 μmol / L.

[0152] The thermal cycler reaction program settings are shown in Table 3 below:

[0153] Table 3 RSV digitalPCR thermal cycle program settings

[0154]

[0155] Data Collection: After amplification, open the top cover and smoothly transfer the chip to the Naica Prism3 for data reading. Start the Crystalreader software and set scanning parameters such as detection channels, exposure time, and sample dilution factor according to the probe fluorophores of different viruses. Run the program to collect data.

[0156] Data analysis: Start CrystalMiner for data analysis to check quality control, fluorescence threshold, and target viral gene copy number.

[0157] Specific results such as Figure 3 As shown in Figure 2, the virus strains showed exponential growth from 12 to 48 hours, and the replication of the two recombinant viruses reached a maximum of 8.0×10 7 cp / μL and 4.7×10 7 cp / μL, the virulence of the two recombinant viruses was weakened compared with the Long-BAC strain in HEp-2 cells, and the replication rate to the peak was slowed down by 12h.

[0158] Example 4 Virus Neutralization Test Determination of Neutralizing Ability of Different Virus Strains to Neutralizing Antibodies

[0159] In the RSV neutralization experiment based on the immunospot method, HEp-2 cells were inoculated in a 96-well plate, cultured to a monolayer, and then mixed with 50 PFU / well of the RSV virus suspension of Example 1 and Nirsevimab monoclonal antibody diluted in different ratios, and incubated at 37°C for 1 hour. The antibody-virus mixture was inoculated into the cell plate, and the unbound virus was removed after adsorption for 1 hour. 1.2% Avicel covering medium was added and cultured for 48 hours. After fixing the cells, they were treated with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% TritonX-100 for 15 minutes, and the spots were detected by immunostaining against RSV F protein (HRP-labeled goat anti-human IgG secondary antibody and TrueBlue staining). The number of spots was counted and the IC50 value of the neutralizing antibody was calculated using a dose-response curve. The results are shown in Figure 2. Figure 4 As shown, the neutralization IC50 of Long-BAC virus and Nirsevimab monoclonal antibody (Nirsevimab) was 5.026 ng / mL, and the neutralization IC50 of Long-BJ1903-AF virus and Long-SY2103-BF virus and Nirsevimab were 1.743 ng / mL and 2.428 ng / mL, respectively, indicating that all three strains showed high sensitivity to Nirsevimab, and the difference in IC50 values ​​was within 3 times, suggesting that the key neutralizing epitope of F protein remained relatively conserved in these strains.

[0160] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A recombinant RSV strain, characterized in that: The recombinant RSV strain comprises an RSV genome or antigenome derived from an RSV Long strain, and the F protein of the Long strain is replaced by the F protein of another RSV genotype strain.

2. The recombinant RSV strain according to claim 1, characterized in that The other RSV genotype strains include the ON1 genotype of RSV A subtype or the BA9 genotype of RSV B subtype.

3. A biomaterial, characterized in that The biomaterial comprises: (1) A vector comprising the gene sequence of the recombinant RSV strain according to any one of claims 1 to 2; (2) A cell, characterized in that the cell comprises the recombinant RSV strain according to any one of claims 1-2 or the vector in (1).

4. The biomaterial according to claim 3, characterized in that The vector includes a BAC vector.

5. A method for constructing a recombinant RSV strain according to any one of claims 1-2, characterized in that: The method comprises replacing the F gene sequence in the RSV Long strain with the F gene sequence of another RSV genotype strain, The method includes: 1) constructing the vector described in claim 4; 2) constructing an auxiliary vector, wherein the auxiliary vector comprises the N, P, L or M2-1 gene; 3) Virus rescue: Using a reverse genetics method, the vector of step 1) and the auxiliary vector of step 2) are co-transfected into cells, and the virus is harvested to obtain a recombinant RSV strain.

6. The method according to claim 5, characterized in that In the step 2), the N, P, L or M2-1 gene includes a codon-optimized N, P, L or M2-1 gene.

7. A reverse genetics system for RSV strains, characterized in that: The reverse genetics system comprises a vector in the biological material according to any one of claims 3 to 4, and an auxiliary vector, wherein the auxiliary vector comprises the N, P, L or M2-1 gene. Preferably, the auxiliary vector comprises a codon-optimized N, P, L or M2-1 gene.

8. Provide a use of the recombinant RSV strain according to any one of claims 1-2, the biological material according to any one of claims 3-4, the recombinant RSV strain obtained by the construction method according to claim 5 or 6, or the reverse genetics system according to claim 7, wherein the use comprises any one of the following: (1) Application in the preparation or screening of products for detecting, preventing and / or treating diseases caused by RSV infection; (2) Application in the preparation or screening of products for detecting and inducing immune responses to RSV antigens.

9. A method for evaluating RSV neutralizing antibodies, characterized in that: The method comprises incubating the recombinant RSV strain according to any one of claims 1-2 or the recombinant RSV strain obtained by the construction method according to claim 5 or 6 with the antibody to be tested and then conducting a neutralization assay.

10. A reagent, characterized in that The reagent includes the recombinant RSV strain according to any one of claims 1-2, the biological material according to any one of claims 3-4, or the reverse genetic system according to claim 7.

Images