Coding nucleotide sequence of cupular virus capsid protein with codon optimization and use thereof
A nucleotide sequence and codon optimization technology, applied in applications, viral peptides, biochemical equipment and methods, etc., can solve problems such as unsatisfactory yields, limited applications, and production limitations
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Embodiment 1
[0041] Example 1 Optimization of the codon of the Norovirus capsid protein coding gene and its expression in insect cells
[0042] The content of this example is to optimize the codon design of the Noro virus capsid protein coding gene, artificially synthesize the optimized gene and use the baculovirus as a carrier to express the codon-optimized Noro virus GGII1, GGII3, GGII4 and GGII7-type capsid protein genes, and their expression levels were compared with wild-type Noro virus capsid protein genes to evaluate the effect of codon optimization. Specific steps are as follows:
[0043] 1. Screening of Calicivirus Positive Diarrhea Stool Specimens
[0044] (1) Specimen processing
[0045] The diarrhea stool specimen was made into a 20% suspension with normal saline, after repeated freezing and thawing twice, centrifuged at 8000rpm for 10min, the supernatant was taken, and immediately used for viral RNA extraction or stored at -80°C for later use.
[0046] (2) Extraction of vir...
Embodiment 2
[0090] Example 2 Code optimization Noro virus capsid protein coding gene expression in mammalian cells is improved
[0091] In order to verify whether the Noro virus capsid protein gene optimized according to the preferred codons used by insect cells can also increase its expression in mammalian cells, in this example, we used adenovirus as a vector to express the codon-optimized Noro virus Virus GGII1, GGII3, GGII4, GGII7 and wild-type GGII3 capsid protein genes were detected in human embryonic kidney epithelial cells 293 and their differences in expression levels. Specific steps are as follows:
[0092] 1. Construction of recombinant adenoviral vector
[0093] (1) Cloning the codon-optimized capsid protein gene into the shuttle vector pShuttle-CMV
[0094] Digest the shuttle plasmid pShuttle-CMV (Stratagen, USA) with restriction endonucleases Bgl II and XhoI: pShuttle-CMV 10 μl, BamH I and XhoI 1 μl each, 10× buffer 2 μl, H 2 O 6 μl. Incubate at 37°C for 2 hours, and rec...
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