Rhabdovirus with recombination superoxide dismutase gene and preparation and application thereof
A technology of recombinant baculovirus and superoxide, applied in the direction of plant gene improvement, application, genetic engineering, etc., can solve the problems of animal virus cross-infection, low safety, etc., to avoid cross-infection, simplify the process flow, and increase expression efficiency high effect
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Embodiment 1
[0031] The preparation of embodiment 1.SOD gene
[0032] According to the published full sequence of related homologous genes, design upstream primer P1 and downstream primer P2, respectively containing BamH I restriction site, start codon ATG and EcoR I restriction site, stop codon TAA, cDNA as template, Primers P1 and P2 were designed as follows:
[0033] P1: 5'-CGGATCCATGCGTGGGGCAAGCACGGA-3' (as shown in SEQ NO.2)
[0034] P2: 5'-ATTTGCGGCCGCTTTAAAACGGCTGCCAAC-3 (as shown in SEQ NO.3)
[0035] The SOD gene of the target fragment was amplified from the genome of Geobacillus kaustophilus HTA426.
Embodiment 2
[0036] Example 2. Construction of the recombinant transfer vector pVL1393-SOD.
[0037] After the amplified target gene SOD is digested by BamH I and EcoR I, it is connected with the transfer vector pVL1393 (purchased from Introvigen) by the method of sticky ends connected by BamH I and EcoR I to obtain the recombinant Transfer vector pVL1393-SOD.
Embodiment 3
[0038] Embodiment 3. Preparation contains the recombinant baculovirus of thermophile SOD gene
[0039] Take 5 μl of the recombinant transfer vector plasmid and 20 μl of linearized viral DNA digested with Bsu36I, make up the total volume to 50 μl with HBS, and mix well. Take 10 μL of liposomes, make up the total volume to 50 μl with HBS, mix well, and mix the two evenly. The supernatant of the adherent cells in the culture flask was aspirated, and the previously cultured BmN cells (purchased from Shanghai Institute of Biochemical Cells) were washed twice with serum-free medium TC-100, and 100 μl of the mixture was added. After continuing to culture at 27°C for 4-6 days, transfer the culture medium of the co-transfected cells to another bottle of well-growing cells. After the infected cells were ruptured, the supernatant was collected and subjected to plaque screening, and the recombinant virus BacPAK-SOD was obtained if there were recombinant plaques. Taking parental virus Bm...
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