Method for wrapping specific double-stranded RNA through prawn virus IHHNV virus-like particles
A wrapping and specific technology, applied in the field of shrimp infectious subcutaneous and hematopoietic tissue necrosis virus, can solve the problems of unreported genome sequence and the impractical clinical application of the injection method.
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Embodiment 1d
[0033] Cloning of embodiment 1 dsRNA expression plasmid
[0034] Artificially synthesize a DNA sequence with a special structure by chemical gene synthesis, such as figure 1 As shown, the sequence sequence is: T7 terminator, T7 promoter, partial sequence of gene vp32 of WSSV virus, T7 promoter and capsid protein binding DNA sequence. This chemically synthesized sequence was initially inserted between the BamHI and XhoI sites in the pBlueScriptIISK(+) vector. In order to achieve co-expression, select the pET-28a(+) vector with the kna resistance gene, and select the homologous enzyme BglII site and XhoI site that have the same sticky end as BamHI on the vector for insertion.
[0035] (1) Restriction endonuclease double digestion: use 20 μl of restriction endonuclease (TAKARA company) enzyme digestion system gene fragment digestion:
[0036]
[0037] Vector digestion:
[0038]
[0039] (2) Carry out gel electrophoresis of the digested products, and cut off the electroph...
Embodiment 2
[0047] Co-expression of embodiment 2 dsRNA expression plasmid and protein expression plasmid
[0048] By co-expressing dsRNA expression plasmid pET-28-vp32b-CBS and protein expression plasmid pET-His-CP, recombinant prokaryotic cells containing dsRNA expression plasmid and protein expression plasmid were obtained, and VLPs encapsulated with dsRNA were obtained through prokaryotic expression .
[0049](1) Transformed cells:
[0050] The dsRNA expression plasmid pET-28-vp32b-CBS obtained in Example 1 and the pET-His-CP protein expression plasmid were used to co-transform Escherichia coli BL21 competent cells. For ampicillin and kanamycin resistance screening, a single colony was picked and transferred to 5 ml LB medium (containing ampicillin 100 μg / ml and kanamycin 100 μg / mL), and cultured overnight at 37°C. The overnight culture was transferred to 200ml LB medium (containing ampicillin 100 μg / ml and kanamycin 100 μg / mL) at a ratio of 1:100, and cultured with shaking at 30°C u...
Embodiment 3
[0063] The condition exploration of embodiment 3 nuclease restriction system
[0064] In order to verify the ability of VLPs to encapsulate specific dsRNA, the nucleic acid encapsulated in VLPs needs to be digested with DNase and RNaseA after extraction to remove DNA and ssRNA. Excessive nuclease or too long enzyme digestion time will also cause dsRNA to be digested, so the appropriate enzyme digestion system needs to be determined by enzyme digestion experiments. The vp32-DNA was amplified by PCR and gel purified. Using vp32-DNA as a template, through the RNA transcription kit T7RiboMAX TM ExpressRNAiSystem (Promega) synthesized vp32-ssRNA and vp32-dsRNA. 100ng of vp32-DNA, vp32-ssRNA and vp32-dsRNA were respectively taken for nuclease digestion. In order to verify the digestion effect, the digested samples were subjected to northern hybridization with vp32-DIG probe. Finally, the suitable concentrations of DNase and RNaseA were determined to be 0.02U / μ and 0.8μg / ml respec...
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