Bivalent attenuated vaccine capable of resisting both papaya leaf distortion mosaic virus (PLDMV) and papaya ringspot virus (PRSV)
A papaya and vaccine technology, applied in the direction of viruses, viral peptides, viruses/phages, etc., can solve problems such as diseases, and achieve a significant effect of disease resistance
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[0049] 1. Cloning of PRSV Nib (PR) fragment:
[0050] Using the designed primer pair PR-F / R (see the table below for the primer sequence), use the PRSV invasive cloning plasmid pG-H (Tuo Decai, 2011) as a template, and use Prime STAR Max Premix (2×) high-fidelity DNA polymerase (TaKaRa) was amplified to obtain a 330bp NIb gene fragment (PR), which only produced low-abundance vsiRNAs combined with the PLDMV virus silencing suppressor HC-Pro, and was an ideal fragment for targeting viral RNA.
[0051] Table 1 Primer sequences
[0052] Primer name Primer sequence PR-F 5′-CAGTGGTCTCTGTCCAGTCCTGACAAGTTACACGGCAACCTT-3′ PR-R 5′-TGGTCTCAGCAGACCACAAGTGATGACTTCGCTATCTGTGAC-3′
[0053] 2. Construction of bivalent attenuated vaccine pPLDMV-PR vector
[0054] The PR fragment was cloned into PLDMV-E using the Nimble Cloning reaction (the 53rd amino acid K of HC-Pro encoded by the wild-type PLDMV virus was mutated to E (see Tuo D, Zhou P, Zhao G, et al.A doubl...
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