P2-mutated Spike protein trimer variant (P2-mutant,S1/S2 cleavage site(furin cleavage sequence)-mutant,trimerization modified) vectors and SARS-CoV-2(2019nCoV) Pseudotyped virus
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Products list of Spike trimer (P2-mutant, S1/S2 cleavage site (furin cleavage sequence)-mutant, trimerization modified)
SARS-CoV-2 P2-mutated Spike protein trimer variant (P2-mutant, S1/S2 cleavage site（furin cleavage sequence)-mutant, trimerization modified)
The world is in midst of the COVID-19 pandemic caused by SARS-CoV-2 (2019nCoV) infection. The Spike protein (S-protein) of SARS-CoV-2 (2019nCoV) mediates receptor (ACE2) binding and cell entry and is the dominant target of the immune system. Most mutations and deletions of SARS-CoV-2 occur in the coronavirus spike protein.
A deletion H69/V70 in Spike was present in over 3,000 sequences worldwide (2.5% of the available data), and largely in Europe from where most of the sequences in GISAID are derived.
The structural impact of the double deletion was predicted by homology modelling of the spike NTD possessing H69/V70 using SWISS-MODEL. The H69/V70 deletion was predicted to alter the conformation of a protruding loop comprising residues 69-76, with the loop being predicted to be predicted to be pulled in the NTD.
Recently a novel SARS-COV-2 (2019nCOV) lineage, the B.1.1.7 lineage, with serials of site mutation, shows stronger infection ability in the UK. In SARS-COV-2 B.1.1.7 lineage, most mutations and deletions occur in the coronavirus spike protein. These deletions also include SARS-CoV-2 P2-mutated Spike protein trimer variant (P2-mutant, S1/S2 cleavage site（furin cleavage sequence)-mutant,trimerization modified).
GeneMedi pseudotype virus (pseudovirus) of SARS-COV-2 (2019nCOV) P2-mutated Spike protein trimer variant (P2-mutant, S1/S2 cleavage site（furin cleavage sequence)-mutant,trimerization modified)
Taking responsibility to help accelerate the COVID-19 vaccine and therapeutic antibody discovery and development, GeneMedi had developed the pseudotype virus (pseudovirus) of SARS-COV-2 (2019nCOV) P2-mutated Spike protein trimer variant (P2-mutant, S1/S2 cleavage site（furin cleavage sequence)-mutant,trimerization modified), which will meet the evaluation of the efficacy of COVID19 vaccines and therapeutic antibodies.
GeneMedi codon-optimized spike mammalian expression vector for SARS-COV-2 (2019nCOV) P2-mutated Spike protein trimer variant (P2-mutant, S1/S2 cleavage site（furin cleavage sequence)-mutant,trimerization modified)
GeneMedi designed a mammalian expression codon-optimized spike mutation/deletion variant vector for COVID-19 SARS-COV-2 (2019nCOV) P2-mutated Spike protein trimer variant (P2-mutant, S1/S2 cleavage site（furin cleavage sequence)-mutant,trimerization modified)
The mutations and deletions information of GeneMedi’s codon-optimized Spike vector for SARS-COV-2 B.1.1.7 lineage in UK COVID-19 pandemic recently:
|
21765-21770 deletion |
Spike-S1 Subunit |
HV 69-70 deletion (Click to more details about HV 69-70 deletion related products) |
Table 1 | All mutations and deletions occur in COVID-19 SARS-COV-2 (2019nCOV) B.1.1.7 lineage
(Click to more details about B.1.1.7 lineage related products).
| Gene | Nucleotide | Amino acid | |
| ORF1ab | C3267T | T1001I | |
| C5388A | A1708D | ||
| T6954C | I2230T | ||
| 11288-11296 deletion | SGF 3675-3677 deletion | ||
| spike | 21765-21770 deletion | HV 69-70 deletion (Click to more details about HV 69-70 deletion related products) | |
| 21991-21993 deletion | Y144 deletion | ||
| A23063T | N501Y (Click to more details about N501Y related products) | ||
| C23271A | A570D | ||
| C23604A | P681H | ||
| C23709T | T716I | ||
| T24506G | S982A | ||
| G24914C | D1118H | ||
| Orf8 | C27972T | Q27stop | |
| G28048T | R52I | ||
| A28111G | Y73C | ||
| N | 28280 GAT->CTA | D3L | |
| C28977T | S235F |
The Spike protein (S-protein) of SARS-CoV-2 (2019nCoV)
The Spike protein (S-protein) of SARS-CoV-2 (2019nCoV) mediates receptor (ACE2) binding and cell entry and is the dominant target of the immune system. The highly flexible Spike protein (S-protein), with its mobile domains, transitions from closed to open conformations to expose its receptor-binding site and, subsequently, from prefusion to postfusion conformations to mediate fusion P2-mutated Spike protein trimer variant vector (P2-mutant, S1/S2 cleavage site(furin cleavage sequence)-mutant, trimerization modified)
The Spike protein (S-protein) of SARS-CoV-2 (2019nCoV) mediates receptor (ACE2) binding and cell entry and is the dominant target of the immune system. The highly flexible Spike protein (S-protein), with its mobile domains, transitions from closed to open conformations to expose its receptor-binding site and, subsequently, from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. SARS-CoV-2 (2019nCoV) Spike protein (S-protein) derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2.
We designed Spike mutations that allow the production of thermostable, disulfide-bonded Spike-protein trimers that are trapped in the closed, prefusion state. Structures of the disulfide-stabilized and non-disulfide-stabilized proteins reveal distinct closed and locked conformations of the Spike protein trimer.
The thermostable, disulfide-bonded Spike-protein trimers construct we make is:
1) S1/S2 cleavage site (furin cleavage sequence) mutant: Modification of the coding sequence of the multibasic S1/S2 cleavage site (furin cleavage sequence) PRRAR to PGSAS at residues 682–685.
2) P2 mutant: proline substitutions at residues 986 and 987.
3) Trimerization statues: add exogenous trimerization signal peptides.
It has been demonstrated that the designed, thermostable, closed Spike trimer can be used in serological assays in COVID-19 test. This trimer protein has potential applications as a reagent for serology, virology and as an immunogen of COVID-19 related diagnostics and vaccine development.
Summary
We designed one upon described thermostable, disulfide-bonded Spike vector, which was used to transiently transfect cells to express ectodomains of the SARS-CoV-2 Spike protein for secretion into cell culture media.
The characters of this Spike vector is as descrpted:
1) S1/S2 cleavage site (furin cleavage sequence) mutant: Modification of the coding sequence of the multibasic S1/S2 cleavage site (furin cleavage sequence) PRRAR to PGSAS at residues 682–685.
2) P2 mutant: proline substitutions at residues 986 and 987.
3) Trimerization statues: add exogenous trimerization signal peptides.
GeneMedi offers pre-made gene ORF mutation plasmids of Spike trimer mutation(thermostable, disulfide-bonded Spike-protein trimers).
The pre-made gene ORF vector of Spike trimer mutation is Codon Optimized for mamamlian and can be used for mammalian expression.
GeneMedi also provides pre-made the lentivirus, adenovirus and AAV vector for the gene ORF plasmids of 2019 nCoV (SARS2 coronavirus).
Reference:
Xiong X, Qu K, Ciazynska KA, Hosmillo M, Carter AP, Ebrahimi S, Ke Z, Scheres SHW, Bergamaschi L, Grice GL, Zhang Y; CITIID-NIHR COVID-19 BioResource Collaboration, Nathan JA, Baker S, James LC, Baxendale HE, Goodfellow I, Doffinger R, Briggs JAG. A thermostable, closed SARS-CoV-2 spike protein trimer. Nat Struct Mol Biol. 2020 Oct;27(10):934-941. doi: 10.1038/s41594-020-0478-5. Epub 2020 Jul 31. PMID: 32737467; PMCID: PMC7116388.
