Nucleic acid molecule encoding structural protein of novel coronavirus and novel coronavirus vaccine
A coronavirus and nucleic acid molecule technology, applied in the field of genetic engineering, can solve the problems of low protein expression and difficult vaccine production of new coronary pneumonia, and achieve the effects of high immunogenicity, high yield and stable structure
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Embodiment 1
[0036] Find the sequences of the E gene, M gene, N gene and S gene of the wild-type novel coronavirus through NCBI, and analyze its codon bias, GC content, and CPG island and other parameters. Four genes were codon-optimized, and the optimized and wild-type sequences are as follows:
[0037] The nucleotide sequence of the optimized E gene is shown in SEQ ID NO.1; the nucleotide sequence of the wild-type E gene is shown in SEQ ID NO.5;
[0038] The nucleotide sequence of the optimized M gene is shown in SEQ ID NO.2; the nucleotide sequence of the wild-type M gene is shown in SEQ ID NO.6;
[0039] The nucleotide sequence of the optimized N gene is shown in SEQ ID NO.3; the nucleotide sequence of the wild-type N gene is shown in SEQ ID NO.7;
[0040] The nucleotide sequence of the optimized S gene is shown in SEQ ID NO.4; the nucleotide sequence of the wild-type S gene is shown in SEQ ID NO.8.
Embodiment 2
[0042] The above-mentioned target gene was obtained by gene synthesis, and it was re-cloned into the expression vector PCI (for the vector map, see Figure 6 ), the present invention takes 2 μg of the target gene and carrier respectively, uses two restriction endonucleases Kpn I and Not I to carry out enzyme digestion at 37°C for 3-4 hours, recovers the gel to obtain the target gene and carrier, and separates the target gene and carrier According to the molar mass of 3:1, use T4 ligase to connect at 16°C for 3-4 hours, then transform it into DH5α strain, pick a single clone and extract the plasmid, and finally identify it by enzyme digestion and Sanger sequencing . Then the recombinant expression plasmids PCI-coE, PCI-coM, PCI-coN and PCI-coS and wild-type PCI-wtE, PCI-wtM, PCI-wtN and PCI-wtS were transfected into HEK293T cells by calcium phosphate transfection and For transient expression, 37°C, 5% CO 2 Cultivate in the environment for 8 hours, replace with fresh DMEM (con...
Embodiment 3
[0045] Preparation of VLPs
[0046] VLPs were transfected with four recombinant expression plasmids PCI-coE, PCI-coM, PCI-coN, PCI-coS or four wild-type plasmids PCI-wtE, PCI-wtM, PCI-wtN, PCI-wtS by calcium phosphate transfection method Mass ratio 1:1:1:1 was co-transfected into HEK293T cells at 37°C, 5% CO 2 Cultivate in the environment for 8 hours, replace with fresh DMEM (containing 10% FBS, 1% double antibody), 37°C, 5% CO 2 Continue culturing for 48 hours, collect the supernatant, centrifuge at 5000rpm, 4°C for 15min, take the supernatant, filter through a 0.45μM filter membrane, ultracentrifuge at 25000rpm, 4°C for 2h 30min, discard the supernatant, and dissolve the precipitate with PBS Finally, the VLPs were concentrated 200 times, and then the optimized (coVLPs) and wild-type (wtVLPs) VLPs yields were compared by methods such as silver staining and western blot. It was found that using the optimized E (small envelope), M (membrane), The yield of VLPs packaged by N (...
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