Recombinant F genotype mumps virus live vector measles vaccine
A technology of mumps virus and mumps virus strain, which is applied in the field of biomedicine and can solve problems such as complexity, impact on vaccine effect, interference, etc.
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[0087] The preparation method of virus strain of the present invention
[0088] In the present invention, a kind of method for preparing recombinant F genotype mumps virus strain is provided, and described method comprises steps:
[0089] (S1) Constructing the full-length recombinant plasmid of the recombinant F genotype mumps virus inserted into the measles virus H gene sequence in the F gene sequence and the HN gene sequence;
[0090] (S2) obtaining three kinds of helper plasmids comprising N gene, P gene and L gene in mumps virus respectively; and
[0091] (S3) Co-transfect the host cells with the full-length recombinant plasmids obtained in (S1) and the three helper plasmids described in (S2), culture for 3 days, lyse the cells, and centrifuge the cell lysate to obtain the supernatant , to obtain the recombinant F genotype mumps virus strain.
[0092] In another preferred embodiment, the host cell is selected from the group consisting of BSR-T7 cells, 293T cells, Vero ce...
Embodiment 1
[0116] Construction of recombinant virus full-length clone
[0117] The basic idea of the full-length cloning of the recombinant virus is as follows: First, based on the F genotype mumps virus reverse genetic manipulation technology platform, the plasmid pMuV (plasmid map as shown in figure 1Shown) linearized, and obtained fragment A of 16405bp by gel recovery; then using the cDNA of MV as a template, primers MV-H-1-F and MV-H-1-R, MV-H-2-F were used respectively and MV-H-2-R, MV-H-3-F and MV-H-3-R amplified to obtain fragment 1, fragment 2 and fragment 3; with primers MV-H-1-F and MV-H- 3-R used fragment 1, fragment 2 and fragment 3 as templates, obtained fragment 4 (3633bp) by fusion PCR, and then digested with restriction endonucleases PmlI and NaeI to obtain fragment 5 with a size of 3370bp; finally fragment A and Fragment 5 was ligated overnight at 16°C with T4 ligase, and transformed into competent cell XL10; finally, positive clones were obtained by enzyme digestion ...
Embodiment 2
[0121] virus rescue
[0122] First, a large number of full-length clones and helper plasmids are extracted with an endotoxin-removing kit; then the cells are seeded in a six-well plate and cultured overnight for transfection, and the confluence of the cells should be 80-90%. The transfection process was as follows: the full-length plasmid pMuV-MV-H (7 μg), the helper plasmid pcDNA3.1-N (1.5 μg), pcDNA3.1-P (0.2 μg), pcDNA3.1-L (1.0 μg) and Lipofectamine TM 2000 transfection reagent (12 μL) was added to 500 μL DMEM medium and mixed gently, and the mixture was incubated at 37°C for 20 min for later use; cells were washed 3 times with PBS, and the supernatant was discarded after the third wash ; Add the mixed solution into the cell well, incubate at 37°C for 6h; then wash with PBS for 3 times, replace with DMEM medium containing 2% serum and 1% antibiotics, and continue to culture at 37°C for 3-4d; put the cells together with the supernatant Freeze and thaw twice, centrifuge a...
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Abstract
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