Preparation method and application of Coxsackie virus B3 virus-like particles
A technology of coxsackie virus and type 3 virus, applied in the direction of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as danger, incomplete inactivation, and disease of the inoculated
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Embodiment 1
[0043] Example 1. Codon optimization and full gene synthesis of CVB3 P1 gene
[0044] The following is a detailed description of the codon optimization of CVB3 P1 gene and the process of full gene synthesis in steps.
[0045] 1. Codon optimization of CVB3 P1 gene
[0046] According to the wild-type CVB3 P1 gene sequence (named CVB3P1W gene, as shown in sequence 6 in the sequence table), after design and repeated verification, an optimized CVB3 P1 gene sequence suitable for expression in yeast cells (named CVB3P1Y gene, such as Sequence 1 is shown in the sequence list), that is, the P1 gene sequence of the CVB3 virus is transformed into a containing such as Sharp PM (Sharp PM, Cowe E. Synonymous Codon Usage in Saccharomyces cerevisiae. Yeast, 1991, 7 ( 7): 657-678.) The optimized sequence of yeast preferred codons described, thereby increasing the expression level of CVB3 VP1, VP2, VP3 and VP4 in the yeast cell culture environment.
[0047] According to the above method, the optimized...
Embodiment 2
[0051] Example 2. Construction of yeast recombinant expression vector carrying CVB3 VP1, VP2, VP3 and VP4 genes
[0052] 1. Preparation of E. coli DH5α competent cells
[0053] Pick a single colony of DH5α (Takara company, catalog number D9057S) from a fresh plate cultured at 37°C for 16-20 hours, inoculate it in 5ml of LB medium without antibiotics, and cultivate overnight at 37°C with vigorous shaking (12-16h) . The next day, transfer 0.5ml from the above culture and transfer it to 50ml LB medium at a volume ratio of 1:100 for about 3 hours. When the OD600 value of the bacterial solution is 3, transfer the bacteria to a sterile medium under aseptic conditions. , Use ice pre-cooled 50ml centrifuge tube, ice bath for 30 minutes. Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, invert the tube for 1 min to drain the remaining culture solution, and use 10 ml of 100 mM CaCl pre-cooled with ice 2 Resuspend the bacterial pellet in the solution in an ice bath for 30 m...
Embodiment 3
[0076] Example 3. Mass expression and purification of recombinant CVB3 virus-like particles in yeast cells
[0077] 1. Preparation of Saccharomyces cerevisiae competent cells
[0078] Pick the monoclonal colony INVSc1 (Saccharomyces cerevisiae, product catalog number: C81000, purchased from Invitrogen) from the Saccharomyces cerevisiae plate, inoculate it in 10ml YPD culture medium (purchased from Beijing Xinjingke Company), shake culture at 30℃ for 16h, take Add a suitable volume of culture to 48ml YPD culture medium and mix to make the OD600 value 0.5. After shaking at 30℃ for 1 hour, the OD600 value is 0.7. After culturing for 30 minutes, transfer the culture to a 50ml sterile centrifuge tube Centrifuge at 1500 rpm for 5 min at room temperature, discard the supernatant, resuspend the pellet with 10ml Sc EasyComp transformation kit (purchased from Invitrogen) in solution I (included in the kit), centrifuge at 1500 rpm at room temperature for 5 minutes, carefully discard the super...
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