Pseudovirus particle capable of being used for evaluating activity of new coronavirus neutralizing antibody and serving as nucleic acid detection standard substance and preparation method of pseudovirus particle

A technology of fake virus particles and viruses, applied in the field of standardization, can solve problems such as biological safety hazards, virus replication safety, hidden dangers, etc.

Pending Publication Date: 2022-02-22
NORTHWESTERN POLYTECHNICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to solve the biological safety hazards existing in the evaluation of the neutralizing antibody effect in the current anti-virus research of the new crown, and the potential safety hazards such as virus replication that may exist in the preparation of the pseudo

Method used

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  • Pseudovirus particle capable of being used for evaluating activity of new coronavirus neutralizing antibody and serving as nucleic acid detection standard substance and preparation method of pseudovirus particle
  • Pseudovirus particle capable of being used for evaluating activity of new coronavirus neutralizing antibody and serving as nucleic acid detection standard substance and preparation method of pseudovirus particle
  • Pseudovirus particle capable of being used for evaluating activity of new coronavirus neutralizing antibody and serving as nucleic acid detection standard substance and preparation method of pseudovirus particle

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The characteristics of the pseudovirion particles prepared by the present invention

[0075] Step S011: Inoculate 293T cells in each well of a six-well culture plate and grow to a monolayer density of 70%;

[0076] Step S012: mix the VSV-G-RBM envelope protein expression plasmid of the present invention, the new coronavirus 7-segment core expression plasmid, and the pMDLg / pRRE and pRSV rev plasmids with liposomes, and transfect 293T cells;

[0077] Step S013: 48 hours after transfection, collect the cell supernatant;

[0078] Step S014: Prepare a six-well cell culture plate, inoculate 293T cells, and grow to a monolayer density of 80%;

[0079] Step S015: inoculate 2 ml of the supernatant harvested in step S013 into each well of the cells in step S014, and continue culturing;

[0080] Step S016: After 48 hours, observe the proportion of fluorescent cells under a fluorescence microscope;

[0081] Step S017: It can be seen that about 60% of the cells can emit green flu...

Embodiment 2

[0088] Embodiment 2 anti-RBM antibody is to the blocking effect of pseudovirion infection Vero cell

[0089]Step S021: 6 BALB / c mice, 3 of which were controls, and the other 3 were intraperitoneally immunized with the new coronavirus S protein, 200ug / mouse / time, a total of 4 times, with an interval of one week between each time. Serum was collected one week after the fourth immunization for use.

[0090] Step S022: Inoculate Vero cells on a 96-well cell culture plate and grow to a monolayer density of 80%;

[0091] Step S023: Add 10ul of mouse immune serum to each well of A1 and A2; add 10ul of normal mouse serum to each well of B1 and B2; add 10ul of normal mouse serum to each well of C1 and C2;

[0092] Step S024: A1, A2, B1, B2, C1, C2, add 20ul of the pseudovirus particles harvested in Step S013 of Example 1 to each well, and continue to cultivate;

[0093] Step S025: observe the number of fluorescent cells in each well under a fluorescence microscope after 48 hours;

...

Embodiment 3

[0095] qPCR and ddPCR quantification of embodiment 3 pseudovirus particles

[0096] Step S031: Extract RNA from viral particles in the supernatant according to the novel coronavirus nucleic acid detection process;

[0097] Step S032: using Oligo dT(16t) as a primer to perform reverse transcription to obtain cDNA;

[0098] Step S032: The pseudovirus particle cDNA containing 861 base RNA of the new coronavirus is subjected to 2-fold serial dilution and quantified by real-time fluorescent quantitative PCR (qPCR) and digital droplet PCR (ddPCR). During the quantification process, SYBER GREEN was used as a fluorescent indicator, and specific primers for different fragments were used to perform real-time fluorescence quantitative PCR (qPCR) amplification and digital droplet PCR (ddPCR) amplification and quantification in parallel.

[0099] The results showed that it was difficult for qPCR to distinguish the 2-fold dilution of the new coronavirus gene template, the lack of regularit...

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Abstract

The invention provides a pseudovirus particle which can be used for evaluating the activity of a new coronavirus neutralizing antibody and used as a nucleic acid detection standard substance and a preparation method of the pseudovirus particle. The problems of biological potential safety hazards caused by the adoption of new crown live viruses for neutralizing antibody effect evaluation in current new crown antiviral research and potential safety hazards such as virus replication possibly existing in preparation of a pseudovirus nucleic acid standard substance for new crown nucleic acid detection are solved. The pseudovirus particle takes lentivirus as a framework; the outer membrane protein is the outer membrane protein fused by a VSV-G protein and a new coronavirus S protein receptor binding motif; the core gene of the gene is an SARS-CoV-2 virus gene segment; the SARS-CoV-2 virus gene segment is a concatemer of seven RNA (Ribonucleic Acid) segments of the virus; the seven RNA fragments are respectively a basic group at the site 13340 to the site 13462, a basic group at the site 15430 to the site 15530, a basic group at the site 22721 to the site 22861, a basic group at the site 26269 to the site 26382, a basic group at the site 28706 to the site 28834, a basic group at the site 28880 to the site 28981 and a basic group at the site 29741 to the site 29890 of the virus gene.

Description

technical field [0001] The invention belongs to the field of standardization technology, the field of virus treatment research and the field of gene detection and diagnosis technology, and specifically relates to the evaluation of the neutralizing activity of a new coronavirus (SARS-CoV-2) antibody, the evaluation of the activity of antiviral drugs, and the qualitative and quantitative analysis of nucleic acid detection Construction and preparation of pseudovirion particles for standard products. Background technique [0002] The new coronavirus is a positive-sense single-stranded RNA virus with an envelope. The particles are round or oval, with a diameter of about 80-120nm. (The S-RBD fragment is 669bp in length); it contains two flanking untranslated regions (UTRs), a 5' long open reading frame (ORF1a / b) and several open reading frames encoding structural proteins. ORF1a / b encodes nonstructural proteins (NSPs) related to viral replication, accounting for two-thirds of the...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/867C12N15/62C12N15/50C12N15/66G01N33/68G01N33/569C12Q1/70C12Q1/6851C12R1/93
CPCC12N7/00C12N15/86C07K14/005G01N33/6854G01N33/56983C12Q1/701C12Q1/6851C12N2770/20021C12N2770/20052C12N2770/20062C12N2740/15043C12N2800/107C12N2770/20022C12N2760/20222C07K2319/00G01N2333/165G01N2469/20C12Q2531/113C12Q2563/159C12Q2563/107Y02A50/30
Inventor 黄庆生田易晓赵雯杨慧邵东燕韩翠翠窦向雅王新月李宇航段苏扬白岳丘
Owner NORTHWESTERN POLYTECHNICAL UNIV
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