Construction and application of pseudotyped VSV (vesicular stomatitis virus) for expressing SARS-CoV-2 spike (S) protein or variant Sdelta21 of SARS-CoV-2 S protein
A spike protein, sars-cov-2 technology, applied in antisense single-stranded RNA virus, positive-sense single-stranded RNA virus, applications, etc., can solve the slow replication speed of coronavirus, limit the use and promotion of neutralizing antibody detection methods and other problems, to achieve the effect of fast replication and easy in vitro amplification
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preparation Embodiment 1
[0051] Preparation Example 1: Optimizing the length of the intracellular segment (cytoplasmic tail, CT) of the SARS-CoV-2S protein so that it can be efficiently embedded in the VSV capsule
[0052] The SARS-CoV-2 virus S gene required by this embodiment will carry out the whole gene synthesis according to the virus strain sequence (GenBankaccession no.MN908947) announced on the NCBI, and the codon of the synthetic gene is optimized according to the human cell codon (SEQID NO.1 ), the sequence was published for the first time, and it has been proved by experiments that the protein can be expressed at a higher level compared with the spike protein S gene sequence of the original new coronavirus strain, which is shown in the fact that we successfully used this sequence to construct Novel COVID-19 vaccine based on recombinant vesicular stomatitis virus. The structure of coronavirus S protein is as follows figure 1 , it is a type I transmembrane protein, including N-terminal signa...
preparation Embodiment 2
[0062] Preparation Example 2: pVSV ΔG -S, pVSV ΔG -S Δ21 Recombinant plasmid construction
[0063] 1. Construct pVSV IND Plasmid, the plasmid clone has the full-length genome sequence of VSV Indiana strain;
[0064] 2. Perform high-fidelity PCR amplification of the S or SΔ21 gene, whose 5' and 3' ends contain MluI and XhoI restriction sites respectively, and clone into pVSV after double digestion IND In the plasmid, make S Δ21 The G gene in VSV is replaced by the gene, and the recombinant plasmid pVSV is obtained ΔG -S or pVSV ΔG -SΔ21, as figure 2 shown.
preparation Embodiment 3
[0065] Preparation Example 3: pVSV MTΔG -S, pVSV MTΔG -SΔ21 recombinant plasmid construction
[0066] 1. Construct pVSV MT Plasmid, its plasmid clone has the genome sequence after the M three site mutation of VSV Indiana strain;
[0067] 2. Perform high-fidelity PCR amplification of the S or SΔ21 gene, whose 5' and 3' ends contain MluI and XhoI restriction sites respectively, and clone into pVSV after double digestion MT In the plasmid, the recombinant plasmid pVSV was obtained MTΔG -S or pVSV MTΔG -SΔ21, as figure 2 shown.
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