COVID-19 pseudovirus as well as preparation method and application thereof

A COVID-19 and COVID-19S technology, applied in the field of COVID-19 pseudovirus and its preparation, can solve the problems of high requirements of the experimental environment, undetectable antagonism, no infectivity, etc., and achieves high sensitivity, Highly infectious and easy to apply effects

Active Publication Date: 2021-03-26
NOVOPROTEIN SCI INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The first method is the closest to reality, but it has high requirements for the experimental environment and cannot be used as a universal detection method; the second method only evaluates the binding inhibition of RBD and ACE2, and cannot detect the antagonism during virus infection ; and the third method overcomes the disadvantages of the first two by using a pseudovirus, because it is not a true virus and does not have the ability to infect, and it can be operated in an ordinary laboratory, simulating the process of virus infection of host cells, compared with the blocking protein The combination of more real
[0005] At present, the pseudovirus coat protein made by most laboratories mainly selects the S protein, and the combination of the S protein and ACE2 is a necessary condition for infection, but the auxiliary role of the M protein and the E protein is ignored in the prior art

Method used

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  • COVID-19 pseudovirus as well as preparation method and application thereof
  • COVID-19 pseudovirus as well as preparation method and application thereof
  • COVID-19 pseudovirus as well as preparation method and application thereof

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preparation example Construction

[0034] The present invention also provides a preparation method of the pseudovirus, the preparation method comprising the following steps: mixing the coat protein particle, the fluorescent reporter plasmid and the helper plasmid, transfecting the host cell, and containing the pseudovirus in the cell supernatant collected after transfection. Viruses, wherein the coat protein particles include a plasmid expressing COVID-19 S protein, a plasmid expressing COVID-19 M protein and a plasmid expressing COVID-19 E protein.

[0035] Before transfection, first mix the plasmids expressing COVID-19 S protein, plasmids expressing COVID-19 M protein and plasmids expressing COVID-19 E protein, and then mix the above mixed plasmids with fluorescent reporter plasmids and helper plasmids. Preferably, the plasmid expressing the COVID-19 S protein, the plasmid expressing the COVID-19 M protein and the plasmid expressing the COVID-19 E protein are mixed in a mass ratio of 5:4:1.

[0036] The host ...

Embodiment 1

[0051] Construction strategy and sequence information of embodiment 1 pseudovirus-related components

[0052] S protein (amino acid sequence shown in SEQ ID NO.1), M protein (amino acid sequence shown in SEQ ID NO.2) and E protein (amino acid sequence shown in SEQ ID NO.3) were respectively constructed into pcDNA3. 3 Vector. pcDNA-S, pcDNA-M, and pcDNA-E plasmids were obtained on TOPO (purchased from Invitrogen, K830001), respectively. The S protein contains the D614G mutation, and the rest of the sites refer to the NCBI reporter sequence. The nucleotides of the mutated S protein The sequence is shown in SEQ ID NO.4. The M protein and E protein refer to the NCBI report sequence (M protein Gene ID: 43740571, E protein Gene ID: 43740570), and the M protein and E protein contain HIS and Flag tags at the C-terminal respectively. The wild-type sequence of EGFP and luciferase (refer to NCBI) was concatenated with 2Asequence and then constructed on the lentiviral vector pLV (purchase...

Embodiment 2

[0054] Embodiment 2 Pseudovirus preparation and quality control

[0055] Pseudovirus preparation

[0056] The constructed plasmids pcDNA-S, pcDNA-M and pcDNA-E were mixed according to the mass ratio (5:4:1) to form S / M / E plasmid (or shell protein plasmid), according to the pMDLg / pRRE (purchased from Addgene, 12251): S / M / E plasmid: pRSV-Rev (purchased from Addgene, 12253): pLV-EGFP-Luc=5:3:2:10 ratio to transfect 293T cells, 72h to collect cell supernatant,- Store at 80°C for later use.

[0057] ELISA detection of pseudovirus coat protein expression

[0058] Coat the plate with 5 μg / ml S protein neutralizing antibody (DA035, Anti-2019-nCoV S-mIgG1 Neutralizing Antibody, Novoprotein), add the prepared pseudovirus suspension, incubate at 37°C for 1 hour, add S pairing protein antibody after washing with PBS (DA041, Anti-2019-nCoV S Antibody, Novoprotein), Anti-HIS-HRP (purchased from Biolegend, 652504) and Anti-Flag-HRP (purchased from GenScript, A01428), incubated at 37°C for...

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Abstract

The invention relates to the technical field of biology, in particular to a COVID-19 pseudovirus as well as a preparation method and application thereof. The COVID-19 pseudovirus is formed by virus packaging of coat protein particles and helper plasmids, wherein the coat protein particles comprise plasmids for expressing a COVID-19 S protein, plasmids for expressing a COVID-19 M protein and plasmids for expressing a COVID-19 E protein. The COVID-19 pseudovirus is packaged by adopting a three-plasmid system, and the S/M/E protein is used for replacing expression a VSV-G protein, so that the COVID-19 pseudovirus is stronger in infection capability and higher in sensitivity compared with the pseudovirus only containing the S protein. Moreover, the COVID-19 pseudovirus carries two fluorescentreporter groups, and different fluorescent reporter groups can be applied to different scenes, so that the COVID-19 pseudovirus is simpler and more convenient to apply.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a COVID-19 pseudovirus and its preparation method and application. Background technique [0002] COVID-19 is a new type of coronavirus that is extremely transmissible. COVID-19 is an enveloped single-stranded RNA virus with an RNA length of nearly 30kb. The envelope of the N protein (Nucleocapsid) shell contains S (Spikeprotein, spike protein), M (Membrane protein, membrane protein) and E (Envelope protein, envelope protein) three proteins. The main infection route discovered so far is that the RBD (Receptor Binding Domain, receptor binding domain) of the S protein of the virus binds to the ACE2 protein on the surface of the cell membrane, resulting in the fusion of the cell membrane and the viral envelope, and finally the virus infects the host cell. [0003] Drug treatment and vaccine prevention are two ways to reduce the harm of the new coronavirus. At present, more than 5 vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/50C12N15/85C12N15/65C12Q1/66G01N21/64
CPCC07K14/005C12N7/00C12N15/65C12N15/85C12N2770/20021C12N2770/20022C12Q1/66G01N21/6428
Inventor 路娜李德彬葛泰根崔利兰张雅婷张杭丁昆雁
Owner NOVOPROTEIN SCI INC
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