CN112375768A - Pseudo-virus of COVID-19 coronavirus, preparation method and application thereof - Google Patents
Pseudo-virus of COVID-19 coronavirus, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a pseudo virus of a COVID-19 coronavirus, a preparation method and application thereof, belonging to the technical field of biological medicines. The pseudovirus is a defective virus genome with a reporter gene, which is a gene involved in the packaging of the defective virus genome with a COVID-19 membrane protein, and may include one or more than one foreign sequence encoding a biologically active substance with or without a reporter gene sequence selected from the group consisting of green fluorescent protein or luciferase, the defective virus genome or a part thereof is derived from VSV, HIV, SARS and other viruses, and one or more structural genes contained therein may be removed or regulated to be inactivated. The pseudovirus can reduce the risk of virus research to the maximum extent, and can be used for screening antiviral drugs, measuring the titer of neutralizing antibodies in infected persons, searching epitopes bound by the neutralizing antibodies on the surface antigen of the COVID-19 coronavirus and evaluating the immune effect of the vaccine.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a pseudo virus of a COVID-19 coronavirus, and a preparation method and application thereof.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is called new coronavirus pneumonia for short, and the world health organization is named as '2019 coronavirus Disease'. Coronavirus (CoV) is a large virus family, and the main causes of infection outbreak of people to date are SARS-CoV, MERS-CoV and SARS-CoV-2, wherein SARS-CoV-2 (also called 2019-nCoV) is a new strain of coronavirus which has never been found in human body before. The virus is a seventh coronavirus capable of infecting people, the incubation period of the infection of the people with 2019-nCoV is generally 1-14 days, the common signs after the infection with the 2019-nCoV comprise respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like, and in more serious cases, the infection can cause pneumonia, severe acute respiratory syndrome, renal failure and even death, and the coronavirus is highly infectious and has higher disease death rate. To study drugs that inhibit the entry of this virus into cells, studies carried out directly with 2019-nCoV are quite dangerous and require harsh experimental conditions.
The pseudovirus has no replication ability, does not cause specific cytopathy caused by 2019-nCoV, and can reduce various risks in the research process of SARS virus to the maximum extent. In addition, the autophagy and infection process of the pseudovirus are the same as those of the euvirus, so that the early process of virus infection can be simulated, and the pseudovirus carries a reporter gene, so that various detections and analyses can be conveniently and quickly carried out, and the method not only can be used for researching the relation between the virus and host cells and cell surface receptors, but also can be used for screening antiviral drugs, measuring the titer of neutralizing antibodies in infected persons, searching epitopes combined with the neutralizing antibodies on 2019-nCoV virus surface antigens, and evaluating the immune effect of the vaccine. Therefore, preparing the pseudo virus of 2019-nCoV and establishing a safe, reliable and efficient in-vitro research infection model of 2019-nCoV infected host cells are safe and effective means for researching infectious disease viruses.
Disclosure of Invention
The first purpose of the invention is to provide a DNA fragment b, the nucleotide sequence of which is shown as SEQ ID NO. 2.
The second purpose of the invention is to provide a recombinant vector containing the DNA fragment b, which is obtained by inserting the optimized DNA fragment b into an expression vector of eukaryotic cells.
The third purpose of the invention is to provide a pseudo-virus of COVID-19 coronavirus, which is a pseudo-virus particle of the COVID-19 coronavirus obtained by the participation of the membrane protein SARS-CoV-2S glycoprotein of the COVID-19 virus in the packaging of a defective virus genome with a reporter gene; wherein:
the reporter gene is selected from green fluorescent protein (EGFP) or luciferase (luciferase);
the defective virus genome or a part thereof is derived from VSV, HIV, SARS and other viruses, and one or more structural genes contained in the defective virus genome can be removed or regulated to be inactivated;
the pseudovirion of the COVID-19 coronavirus may include one or more foreign sequences encoding a biologically active substance, with or without a reporter sequence.
The fourth object of the present invention is to provide a method for preparing the above pseudovirus of COVID-19 coronavirus, comprising:
(1) preparation of plasmid one: inserting a DNA fragment a of a membrane protein SARS-CoV-2S glycoprotein of COVID-19 virus shown as SEQ ID NO. 1 into a eukaryotic cell expression vector containing a Flag label to obtain the recombinant expression vector;
(2) preparation of plasmid two: the DNA fragment b of the membrane protein SARS-CoV-2S glycoprotein of the COVID-19 virus shown as SEQ ID NO. 2 is optimized and inserted into a eukaryotic cell expression vector to obtain the recombinant DNA;
(3) preparation of plasmid three: removing envelope protein genes and defecting structural genes Pol and Gag from a virus genome to enable the virus genome to carry a reporter gene;
(4) and (2) transfecting the plasmid I and the plasmid II with a plasmid III and a lentivirus packaging plasmid respectively by using Polyetherimide (PEI) through a three-plasmid lentivirus packaging system into a packaging cell line HEK293T, collecting cell supernatant after 48-72 hours, and extracting and purifying the cell supernatant to obtain the COVID-19 coronavirus pseudovirions.
According to an embodiment of the method for preparing pseudoviruses of the present invention, the lentiviral packaging plasmid is a psPAX2 plasmid.
According to one embodiment of the pseudovirus preparation method, the eukaryotic cell expression vector is a pCMV3 plasmid.
The fifth purpose of the invention is to provide the application of the pseudo virus of the COVID-19 coronavirus as a detection means in the research of the COVID-19 neutralizing antibody or the drug screening research.
The sixth object of the present invention is to provide a pseudovirus cell model of COVID-19 coronavirus, which is obtained by infecting 293T-hACE2 cells with the above pseudovirus of COVID-19 coronavirus.
The seventh purpose of the invention is to provide the application of the pseudo virus cell model of the COVID-19 coronavirus in screening the inhibitor for inhibiting the entry of the COVID-19 into the cells.
According to one embodiment of the application of the cell model of the pseudo-virus of the COVID-19 coronavirus, the infection efficiency of the pseudo-virus of the COVID-19 coronavirus can be quantitatively determined by examining the expression amount of a reporter gene carried by the defective virus genome expressed in a cell.
An eighth object of the present invention is to provide a method for quantifying pseudovirions of COVID-19 coronavirus, comprising the step of quantifying the S protein content in a culture medium for packaging pseudovirions of COVID-19 coronavirus by sandwich ELISA method using anti-RBD (Sino Biological, 40592-MM57) plating and anti-S1-biotin (Sino Biological, 40591-MM43) binding.
The invention has the beneficial effects that:
1. the pseudo virus of the COVID-19 coronavirus does not have the replication capacity, does not cause specific cytopathy caused by the COVID-19 coronavirus, and can reduce various risks in the virus research process to the maximum extent.
2. The pseudovirus autophagy and infection process of the COVID-19 coronavirus are the same as those of a true virus, the early process of virus infection can be simulated, and the pseudovirus carries a reporter gene, so that various detections and analyses can be conveniently and rapidly carried out, and the COVID-19 coronavirus can be used for researching the relationship between the virus and host cells and cell surface receptors, screening antiviral drugs, measuring the titer of neutralizing antibodies in an infected person, searching epitopes combined with the neutralizing antibodies on 2019-nCoV virus surface antigens and evaluating the immune effect of vaccines.
Drawings
FIG. 1 is a diagram showing a comparison between plasmid one (pCMV 14-3X-Flag-SARS-CoV-2S) and plasmid two (Zhou-COVID-19-Spike) in a preferred embodiment of the present invention.
FIG. 2 is a comparison of luciferase activities produced after infection of 293T-ACE2 cells with three pseudoviruses packaged with luciferase reporter gene plasmid one, plasmid two and Vesicular Stomatitis Virus Glycoprotein (VSVG) in a preferred embodiment of the invention.
FIG. 3 is a diagram showing the comparison of RNA virus copy number of plasmid one and plasmid-packaged pseudoviruses for S protein quantification by RT-PCR in a preferred embodiment of the present invention.
FIG. 4 is a diagram showing the quantitative comparison of the S protein content of plasmid-I and plasmid-II pseudoviruses by ELISA in a preferred embodiment of the present invention.
Detailed Description
The present invention is further illustrated by the following examples and comparative examples, which are intended to be illustrative only and are not to be construed as limiting the invention. The technical scheme of the invention is to be modified or replaced equivalently without departing from the scope of the technical scheme of the invention, and the technical scheme of the invention is covered by the protection scope of the invention.
The nucleotide sequence of the DNA fragment a provided by the invention is shown in SEQ ID NO. 1.
The invention provides a recombinant vector containing the DNA fragment a, which is obtained by inserting the DNA fragment a into a eukaryotic cell expression vector containing a Flag tag.
The nucleotide sequence of the DNA fragment b provided by the invention is shown in SEQ ID NO. 2.
The invention provides a recombinant vector containing the DNA fragment b, which is obtained by inserting the optimized DNA fragment b into an eukaryotic cell expression vector.
The pseudo virus of the COVID-19 coronavirus provided by the invention is as follows: is a pseudo-virus particle of the COVID-19 coronavirus, which is obtained by the participation of a membrane protein (SARS-CoV-2S glycoprotein) of the COVID-19 virus in the packaging of a defective virus genome with a reporter gene; wherein the reporter gene may be EGFP or Luciferase, the defective viral genome or a part thereof may be derived from VSV, HIV, SARS and other viruses, one or more structural genes contained in the defective viral genome may be deleted or may be regulated to be inactivated, and the pseudo-viral particle of the COVID-19 coronavirus may include one or more foreign sequences encoding a biologically active substance, with or without the reporter gene sequence.
The surface of the pseudovirus membrane protein of the COVID-19 coronavirus provided by the invention is provided with the COVID-19 membrane protein, and the capsular sac is internally provided with defective genome and structural protein of other viruses with reporter genes. The infection efficiency of the pseudovirus can be quantitatively judged by infecting COVID-19 susceptible cells such as 293T-hACE2 with the pseudovirus particles of the COVID-19 coronavirus, expressing a reporter gene carried by a defective virus genome in the cells and detecting the expression level of the reporter gene.
Experiments prove that the neutralizing antibody in serum of a mouse immunized by the S protein trimer and the anti-S1 and anti-RBD monoclonal neutralizing antibody can effectively block the infection of the pseudovirus of the COVID-19 coronavirus on target cells, so that the expression level of luciferase in the target cells is obviously reduced. The pseudovirus can be used as an effective detection means for researching a COVID-19 neutralizing antibody.
The preparation method of the pseudo virus of the COVID-19 coronavirus provided by the invention comprises the following steps:
(1) preparation of plasmid one (pCMV 14-3X-Flag-SARS-CoV-2S): inserting DNA fragment a (shown as SEQ ID NO: 1) of COVID-19 membrane protein (SARS-CoV-2S glycoprotein) into eukaryotic cell expression vector containing Flag label;
(2) preparation of plasmid two (Zhou-COVID-19-Spike): inserting the DNA fragment b (shown as SEQ ID NO: 2) of the COVID-19 membrane protein into a eukaryotic cell expression vector after optimization;
(3) preparation of plasmid III (Zhou-pLOV-GFP-Luc): the envelope protein gene in the viral genome is removed and the structural genes Gag and Pol are deficient, so that the viral genome carries the reporter genes EGFP and luciferase.
(4) Transfecting the plasmid I and the plasmid II with a plasmid III containing a defective other virus genome carrying a reporter gene and a lentivirus packaging plasmid psPAX2 (plasmid IV) respectively by using polyetherimide through a three-plasmid lentivirus packaging system into a packaging cell line HEK293T, collecting cell supernatant after 48-72 hours, and extracting and purifying the obtained pseudo virus particles of the COVID-19 coronavirus from the cell supernatant; among them, the virus supernatant can be concentrated using a Lenti-X Concentrator (Clontech, 631231) virus concentration kit.
In the embodiment of the invention for preparing the pseudo-virus of the COVID-19 coronavirus, a PEI transfection method is adopted to co-transfer a plasmid containing a regulated COVID-19 virus envelope protein gene sequence and other plasmids in a lentivirus three-plasmid packaging system into a packaging cell line HEK293T, each 10cm dish contains 9 mu g of plov-EGFP-Luc, 6 mu g of psPAX2 and 3 mu g of a plasmid expressing SARS-CoV-2S glycoprotein, so that the packaging cell line produces the virus, and the harvested virus particles are the pseudo-virus particles expressing the SARS-CoV-2S glycoprotein. Wherein, the envelope protein of COVID-19 is involved in the virus with reporter genes EGFP and luciferase.
The following is further illustrated by specific examples.
EXAMPLE 1 construction of plasmid I and plasmid II of COVID-19S protein expression vector
The SARS-CoV-2S glycoprotein gene sequence MN908947.3 of the expression membrane protein in NCBI is inquired:
plasmid one: the C-terminal 19 amino acids of S-glycoprotein were removed.
And a second plasmid: the signal peptide (GSP) of the VSVG protein is connected to the amino acid 16aa corresponding to the signal peptide (GSP) of the VSVG protein before the sequence of the amino acid 14-1213 corresponding to the extracellular region (SARSS) of the COVID-19S protein, and the transmembrane region and the intracellular region (G TMC) of the VSVG protein are connected to the signal peptide after codon optimization, and then are subjected to gene synthesis by Beijing Yinqiao science and technology Limited company and are connected to the eukaryotic cell expression vector pCMV3 in a homologous recombination mode, as shown in FIG. 1.
Example 2 pseudovirus preparation and concentration
Transfecting a plasmid I and a plasmid II containing SARS-CoV-2S glycoprotein membrane proteins, a plasmid III containing defective other virus genomes carrying reporter genes and a lentivirus packaging plasmid (plasmid IV) into a packaging cell line HEK293T by a three-plasmid lentivirus packaging system by adopting polyetherimide, collecting cell supernatants after 48-72 hours, concentrating the virus supernatants by a Lenti-X Concentrator, and extracting and purifying the pseudovirus particles of the obtained COVID-19 coronavirus from the cell supernatants.
Example 3 identification of pseudoviruses
50 microliters of a concentrate of pseudovirus containing COVID-19 coronavirus were infected with the known COVID-19 susceptible 293T-hACE2 and non-susceptible 293T cells in 96-well plates at 30,000 cells per well. And (3) removing the culture medium after 12h of infection, replacing the culture medium with a DMEM fresh complete culture medium, continuing to culture at 37 ℃ for 48h, detecting the expression quantity of EGFP on the surface of the cell by flow cytometry, and further identifying the infection efficiency of the pseudovirus by detecting the expression of luciferase.
As shown in FIG. 2, the pseudovirion can well infect susceptible cell 293T-hACE2, and the optimized plasmid infection efficiency is obviously improved and is 10 times higher than that of the non-optimized S protein vector. The pseudovirus is more effectively mediated by the plasmid-two-package pseudovirus than by the plasmid-one-package pseudovirus, and the packaged culture solution can be directly used for a pseudovirus infection model without concentration.
As shown in FIG. 3, the quantitative analysis of the virus copy number by qRT-PCR (Clontech, 631235) revealed that the difference in expression of the pseudovirus reporter gene luciferase was not due to the difference in the amount of replication of the pseudovirus RNA.
As shown in FIG. 4, the S protein content in the virus packaging medium was quantified by sandwich ELISA, which was plated with anti-RBD (Sino Biological, 40592-MM57) and anti-S1-biotin (Sino Biological, 40591-MM43) and calibrated with S-trimer of known concentration. The results show that the S-trimer concentrations in the pseudovirus-containing supernatants produced by the optimized plasmid and the non-optimized plasmid under the same experimental conditions are different by about 7.2 times, which indicates that plasmid two mediates the generation of more pseudovirus particles.
EXAMPLE 4 detection of neutralizing antibody Activity
Preparation of S protein trimer vaccine: the S protein coding region Cys15-Gln1208(Genbank accession number MN908947) and 6x histag are fused, expressed in CHO cells, and purified by affinity chromatography to reach 95% purity.
Preparation of protein S unimer vaccine: the S protein coding region Met1-Gln1224(Genbank access number MN908947) is fused with 9x histag, expressed in insect cells and purified by affinity chromatography to reach 95% purity.
C57B6 mouse immunization: C57B6 male mice at 6 to 8 weeks were immunized by intramuscular injection at a schedule of 0, 7, 14 days, each time with 5ug of S protein, poii: the C adjuvant 50ug was diluted to 100ul with PBS and injected intramuscularly in the mouse hip, and 7 days after the last immunization, tail vein blood was collected from the mouse to determine the serum neutralizing antibody titer.
The activity of the neutralizing antibody was detected using a pseudovirus of COVID-19 coronavirus. Susceptibility to pseudovirusesCell 293T-hACE2 at a density of 3X 104Inoculating each cell in a 96-well flat bottom plate to allow the cells to adhere to the wall; diluting serum according to 5-fold gradient to set concentration gradient, mixing 50 mu L of diluent with isovolumetric pseudovirus, incubating at 37 ℃ for 1h, adding into a cell plate prepared in advance, culturing at 37 ℃ for 12h, changing the culture solution into a fresh culture medium, continuously culturing for 48h, and detecting luciferase.
The result shows that the serum of the immunized mouse has COVID-19 neutralizing antibody and can effectively block the infection of the pseudovirus of the COVID-19 coronavirus to target cells, so that the RLU value is obviously reduced. After immunization with the S protein trimer, the average titer of the mouse serum neutralizing antibody reaches 1:3000, which indicates that the neutralizing antibody titer is remarkably higher than that of the S protein monomomer vaccine.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Sequence listing
<110> university of Tongji
Pseudo-virus of <120> COVID-19 coronavirus and preparation method and application thereof
<141> 2020-11-16
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3783
<212> DNA
<213> unknown ()
<400> 1
atgaaatgtc ttctctattt ggcgttcctg tttatcggag tgaactgtca gtgtgtgaac 60
ctgaccacca ggacccaact tcctcctgcc tacaccaact ccttcaccag gggagtctac 120
taccctgaca aggtgttcag gtcctctgtg ctgcacagca cccaggacct gttcctgcca 180
ttcttcagca atgtgacctg gttccatgcc atccatgtgt ctggcaccaa tggcaccaag 240
aggtttgaca accctgtgct gccattcaat gatggagtct actttgccag cacagagaag 300
agcaacatca tcaggggctg gatttttggc accaccctgg acagcaagac ccagtccctg 360
ctgattgtga acaatgccac caatgtggtg attaaggtgt gtgagttcca gttctgtaat 420
gacccattcc tgggagtcta ctaccacaag aacaacaagt cctggatgga gtctgagttc 480
agggtctact cctctgccaa caactgtacc tttgaatatg tgagccaacc attcctgatg 540
gacttggagg gcaagcaggg caacttcaag aacctgaggg agtttgtgtt caagaacatt 600
gatggctact tcaagattta cagcaaacac acaccaatca acctggtgag ggacctgcca 660
cagggcttct ctgccttgga accactggtg gacctgccaa ttggcatcaa catcaccagg 720
ttccagaccc tgctggctct gcacaggtcc tacctgacac ctggagactc ctcctctggc 780
tggacagcag gagcagcagc ctactatgtg ggctacctcc aaccaaggac cttcctgctg 840
aaatacaatg agaatggcac catcacagat gctgtggact gtgccctgga cccactgtct 900
gagaccaagt gtaccctgaa atccttcaca gtggagaagg gcatctacca gaccagcaac 960
ttcagggtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 1020
tttggagagg tgttcaatgc caccaggttt gcctctgtct atgcctggaa caggaagagg 1080
attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 1140
aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 1200
gactcctttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 1260
attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 1320
agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 1380
aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 1440
acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 1500
caaccaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 1560
ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 1620
tgtgtgaact tcaacttcaa tggactgaca ggcacaggag tgctgacaga gagcaacaag 1680
aagttcctgc cattccaaca gtttggcagg gacattgctg acaccacaga tgctgtgagg 1740
gacccacaga ccttggagat tctggacatc acaccatgtt cctttggagg agtgtctgtg 1800
attacacctg gcaccaacac cagcaaccag gtggctgtgc tctaccagga tgtgaactgt 1860
actgaggtgc ctgtggctat ccatgctgac caacttacac caacctggag ggtctacagc 1920
acaggcagca atgtgttcca gaccagggct ggctgtctga ttggagcaga gcatgtgaac 1980
aactcctatg agtgtgacat cccaattgga gcaggcatct gtgcctccta ccagacccag 2040
accaacagcc caaggagggc aaggtctgtg gcaagccaga gcatcattgc ctacacaatg 2100
agtctgggag cagagaactc tgtggcttac agcaacaaca gcattgccat cccaaccaac 2160
ttcaccatct ctgtgaccac agagattctg cctgtgagta tgaccaagac ctctgtggac 2220
tgtacaatgt atatctgtgg agacagcaca gagtgtagca acctgctgct ccaatatggc 2280
tccttctgta cccaacttaa cagggctctg acaggcattg ctgtggaaca ggacaagaac 2340
acccaggagg tgtttgccca ggtgaagcag atttacaaga cacctccaat caaggacttt 2400
ggaggcttca acttcagcca gattctgcct gacccaagca agccaagcaa gaggtccttc 2460
attgaggacc tgctgttcaa caaggtgacc ctggctgatg ctggcttcat caagcaatat 2520
ggagactgtc tgggagacat tgctgccagg gacctgattt gtgcccagaa gttcaatgga 2580
ctgacagtgc tgcctccact gctgacagat gagatgattg cccaatacac ctctgccctg 2640
ctggctggca ccatcacctc tggctggacc tttggagcag gagcagccct ccaaatccca 2700
tttgctatgc agatggctta caggttcaat ggcattggag tgacccagaa tgtgctctat 2760
gagaaccaga aactgattgc caaccagttc aactctgcca ttggcaagat tcaggactcc 2820
ctgtccagca cagcctctgc cctgggcaaa ctccaagatg tggtgaacca gaatgcccag 2880
gctctgaaca ccctggtgaa gcaactttcc agcaactttg gagccatctc ctctgtgctg 2940
aatgacatcc tgagcagact ggacaaggtg gaggctgagg tccagattga cagactgatt 3000
acaggcagac tccaatccct ccaaacctat gtgacccaac aacttatcag ggctgctgag 3060
attagggcat ctgccaacct ggctgccacc aagatgagtg agtgtgtgct gggacaaagc 3120
aagagggtgg acttctgtgg caagggctac cacctgatga gttttccaca gtctgcccct 3180
catggagtgg tgttcctgca tgtgacctat gtgcctgccc aggagaagaa cttcaccaca 3240
gcccctgcca tctgccatga tggcaaggct cactttccaa gggagggagt gtttgtgagc 3300
aatggcaccc actggtttgt gacccagagg aacttctatg aaccacagat tatcaccaca 3360
gacaacacct ttgtgtctgg caactgtgat gtggtgattg gcattgtgaa caacacagtc 3420
tatgacccac tccaacctga actggactcc ttcaaggagg aactggacaa atacttcaag 3480
aaccacacca gccctgatgt ggacctggga gacatctctg gcatcaatgc ctctgtggtg 3540
aacatccaga aggagattga cagactgaat gaggtggcta agaacctgaa tgagtccctg 3600
attgacctcc aagaactggg caaatatgaa caatacatca agtggccatt tttcttcatt 3660
attgggctca tcatcggcct gtttctggtg ttgagggtgg gaatccacct ctgcatcaaa 3720
ctgaagcaca caaaaaaaag acagatctat acagacatcg agatgaaccg gctgggcaag 3780
taa 3783
<210> 2
<211> 3778
<212> DNA
<213> unknown ()
<400> 2
aagcttcacc atgttcgttt tccttgttct gttgcctctc gttagtagcc aatgcgtcaa 60
ccttactact agaacccagc tccctccagc atataccaac tctttcacca ggggcgtata 120
ttacccggac aaagtgttcc gctcaagtgt gctgcattct acgcaggacc ttttcttgcc 180
ctttttcagt aatgttactt ggtttcatgc tatccatgtg tctggaacta acggaaccaa 240
gcgctttgac aaccccgtcc tccctttcaa cgatggcgtg tacttcgctt ccacggaaaa 300
gtcaaacata attcgcggct ggatctttgg tacaacactc gactcaaaga cgcagagcct 360
gctgatcgtt aataacgcta caaatgttgt gataaaggtg tgtgaatttc agttctgcaa 420
tgatcccttc ctgggtgtgt actaccataa gaataacaag agctggatgg aatccgaatt 480
tagggtttac agttccgcta acaactgcac attcgaatac gtaagccagc catttcttat 540
ggatcttgag ggcaagcaag gaaacttcaa gaacttgagg gagttcgtgt tcaaaaatat 600
cgacggctat tttaagatat atagcaagca cactccaata aacttggtgc gcgacctgcc 660
ccagggattc tctgctctgg agcccctggt ggatctgccc attggaataa acataactcg 720
ctttcaaaca ctgctcgccc tgcatcgcag ttacctcacc cctggtgata gtagttcagg 780
atggacagca ggagccgccg catactacgt cggctacctg cagcctagga ccttcttgct 840
gaagtacaac gagaacggta caataactga cgctgtggac tgcgctctgg accctctgtc 900
cgagacgaag tgcaccctga agagctttac tgttgaaaaa ggcatttacc aaaccagcaa 960
cttccgcgtc cagccaaccg agagcatcgt cagatttccc aacattacaa atctgtgtcc 1020
cttcggcgag gtgttcaacg ccacacgctt cgcttcagtg tacgcatgga accgcaagcg 1080
catatctaac tgcgtcgcgg attattctgt cctctacaac tccgcctctt tctccacctt 1140
caagtgctac ggagtgtcac cgactaagct gaacgatctc tgctttacca acgtctacgc 1200
ggactccttc gtgataagag gtgatgaagt gagacaaata gccccaggtc agactggtaa 1260
gatcgcagat tacaactaca aattgcctga tgatttcact ggttgcgtta tcgcgtggaa 1320
ctctaataac ctcgattcta aggtcggtgg taactacaat tacctgtacc gcttgtttag 1380
gaagtcaaac ctgaagcctt tcgagaggga tatttcaacc gaaatctatc aagcgggttc 1440
aacaccgtgt aacggtgtgg aaggatttaa ctgctacttc cccctgcagt cttacggatt 1500
ccagccaacc aatggcgtgg gttaccaacc ttatcgcgtg gtggttctga gtttcgaact 1560
gttgcacgct cccgccacgg tatgcggtcc caagaagagc actaacttgg tgaagaataa 1620
gtgcgtgaat ttcaatttca atggcctcac tggaactgga gtgctgaccg aatccaataa 1680
gaagttcttg cccttccagc agttcggaag agacattgct gacacaaccg acgcggtgcg 1740
cgatcctcag actctggaga tattggacat tacaccatgt tctttcggcg gtgtgtctgt 1800
cattactccg ggcacgaata ctagcaacca ggtagccgtg ctgtaccaag acgtgaattg 1860
cacagaggtt cccgtcgcaa ttcacgctga ccagctgacc cccacgtgga gggtttacag 1920
cactggtagt aacgtcttcc agacgagagc cggttgcttg atcggagcgg aacatgtgaa 1980
taactcctac gagtgcgaca tccccatcgg agccggtata tgcgcctctt atcagacaca 2040
aactaactca cccaggagag cccgcagtgt ggcttctcaa agcattatag catacactat 2100
gtctcttggt gccgaaaatt ccgtggccta ttctaacaat tcaatcgcca tcccaaccaa 2160
cttcacaatt agcgtgacta ccgaaatact gcctgtgagc atgacgaaaa ccagcgtaga 2220
ctgcactatg tatatctgtg gagactccac tgagtgctcc aaccttctcc tgcagtacgg 2280
tagcttctgt acccaattga accgcgccct tacaggcatc gctgttgagc aagataagaa 2340
tacccaggaa gtttttgccc aggttaagca gatatacaaa acaccgccca ttaaggactt 2400
cggaggcttc aacttctctc agatactgcc tgacccctcc aagccatcaa aacgcagctt 2460
cattgaggac ctcttgttca acaaagtgac tctggctgat gctggcttca ttaagcagta 2520
cggagattgc ctgggagata ttgctgccag ggacctcatc tgcgcccaga agtttaatgg 2580
cctgacagtc ttgcccccac ttctgacaga cgagatgatt gctcagtaca catctgccct 2640
cctcgctggc accataacat ccggatggac atttggtgct ggtgctgccc tccagattcc 2700
cttcgcaatg cagatggcgt atcgctttaa cggcatcggt gtcacacaaa acgtgttgta 2760
tgagaaccaa aagctcatcg ctaaccagtt taattctgct attggtaaga ttcaggacag 2820
cctgtcatca accgcgtctg cccttggtaa gttgcaggac gtggtgaacc agaatgctca 2880
ggctttgaat actctggtga agcaactctc ttcaaatttc ggcgctatct cttctgtgtt 2940
gaacgacatc ctgagtcgcc ttgataaggt ggaagctgaa gttcaaattg atagattgat 3000
tactggcagg ctccagtctt tgcagaccta cgttacacag cagctgatta gggcggctga 3060
aattagagct tccgccaatc tggctgcaac caagatgtcc gaatgcgtcc tgggtcagtc 3120
aaagcgcgtt gacttttgtg gtaaaggcta ccacctcatg tcatttcccc agtcagcacc 3180
tcacggagta gtgttcctcc acgtcaccta cgttccagca caggaaaaga attttaccac 3240
tgcgccggca atctgtcacg acggtaaggc acacttcccc cgcgagggcg tattcgtgtc 3300
taacggaact cattggttcg tcacacagag aaacttctat gagcctcaga tcattaccac 3360
cgacaataca tttgtgtccg gtaactgcga cgttgtgatt ggaatcgtca acaacactgt 3420
gtacgatcca cttcagccag aactggatag cttcaaggaa gaattggaca aatatttcaa 3480
aaatcacact tcacccgatg tggacctggg tgacattagt ggtatcaatg cgtccgtggt 3540
caatattcaa aaagagattg acaggctcaa cgaagtggcc aagaacctga acgaaagtct 3600
tatcgatctg caagaattgg gaaagtatga gcagtacatc aagtggccgt ggtacatttg 3660
gttgggtttt atcgccggtc tgatcgccat cgttatggtt accattatgc tttgctgcat 3720
gacgagctgt tgctcctgtc tgaagggatg ctgctcttgc ggatcatgtt gcggatcc 3778
Claims (10)
1. A DNA fragment b, the nucleotide sequence of which is shown in SEQ ID NO. 2.
2. A recombinant vector comprising the DNA fragment b of claim 1, wherein the DNA fragment b of claim 1 is optimized and inserted into an expression vector of a eukaryotic cell.
3. A pseudo-virus of COVID-19 coronavirus is a pseudo-virus particle of COVID-19 coronavirus obtained by involving a membrane protein SARS-CoV-2S glycoprotein of the COVID-19 virus in the packaging of a defective virus genome having a reporter gene; wherein:
the reporter gene is selected from green fluorescent protein or luciferase;
the defective virus genome or a part thereof is derived from a virus including VSV, HIV, SARS, and one or more structural genes contained in the defective virus genome can be removed or regulated to be inactivated;
the pseudovirion of the COVID-19 coronavirus may include one or more foreign sequences encoding a biologically active substance, with or without a reporter sequence.
4. The method of producing a pseudovirus of the COVID-19 coronavirus of claim 3, comprising the steps of:
(1) preparation of plasmid one: inserting a DNA fragment a of a membrane protein SARS-CoV-2S glycoprotein of COVID-19 virus shown as SEQ ID NO. 1 into a eukaryotic cell expression vector containing a Flag label to obtain the recombinant expression vector;
(2) preparation of plasmid two: the DNA fragment b of the membrane protein SARS-CoV-2S glycoprotein of COVID-19 virus shown as SEQ ID NO. 2 in claim 1 is optimized and inserted into eukaryotic cell expression vector to obtain the product;
(3) preparation of plasmid three: removing the envelope protein gene from a virus genome and deleting the structural genes Pol and Gag so that the virus genome carries the reporter gene;
(4) and (3) transfecting the plasmid I and the plasmid II, the plasmid III and the lentivirus packaging plasmid respectively into a packaging cell line HEK293T by using a three-plasmid lentivirus packaging system through polyetherimide, collecting cell supernatant after 48-72 hours, and extracting and purifying to obtain the COVID-19 coronavirus pseudovirion.
5. The method of claim 4, wherein the lentiviral packaging plasmid is a psPAX2 plasmid, and/or
The eukaryotic cell expression vector is pCMV3 plasmid.
6. Use of the COVID-19 coronavirus pseudovirus of claim 4 in COVID-19 neutralizing antibody studies or drug screening studies.
7. A pseudoviral cell model of COVID-19 coronavirus, which is obtained by infecting 293T-hACE2 cells with the pseudovirus of COVID-19 coronavirus according to claim 4.
8. Use of the pseudo-viral cell model of the COVID-19 coronavirus according to claim 7 for screening for inhibitors that inhibit the entry of COVID-19 into the cell.
9. The use according to claim 8, wherein the infection efficiency of the CoVID-19 coronavirus pseudovirus is determined quantitatively by examining the expression level of a reporter gene carried in the cell expressing a defective virus genome.
10. A method for quantifying COVID-19 coronavirus pseudovirion particles, which is characterized in that the S protein content of a COVID-19 coronavirus pseudovirion packaging culture solution prepared according to claim 4 is quantified by a sandwich ELISA method combining anti-RBD (Sino Biological, 40592-MM57) plating and anti-S1-biotin (Sino Biological, 40591-MM 43).
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