Recombinant mumps virus jeryl lynn 2 based vaccine
A mumps virus and genome technology, applied in the field of new vaccines against mumps, can solve problems such as difficulty in further developing vaccines
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Embodiment 1
[0110] Example 1: Construction of the clone of the full-length DNA genome of the attenuated JL2 strain of mumps virus Production of whole mumps JL2 long cDNA
[0111] Six genome sequence fragments were chemically synthesized (GeneArt-Thermo Fisher Scientific) to cover the total genome length of mumps virus (15,384 bp). Subsequently, each genome fragment and vector was cleaved by specific restriction endonucleases (NewEngland Bio Labs) and cloned into an intermediate vector. through such as figure 1 Each fragment was verified by its separation on an agarose gel as indicated. Fragments obtained after restriction digestion have been described as follows:
[0112] Fragment 1: NotI-RsrII at positions 1 to 3576 (SEQ ID NO.1);
[0113] Fragment 2: RsrII-AvrII at positions 3576 to 6258 (SEQ ID NO.2);
[0114] Fragment 3: AvrII-XhoI at positions 6258 to 8438 (SEQ ID NO.3);
[0115] Fragment 4: XhoI-KpnI at positions 8438 to 10498 (SEQ ID NO.4);
[0116] Fragment 5: KpnI-XmaI ...
Embodiment 2
[0121] Example 2: Rescue of attenuated mumps Jeryl-Lynn 2 virus
[0122] by pMuV JL2 with helper plasmid p_NJL2 , p_p JL2 , p_L JL2 Commercially available HEK 293T cells were transfected together with pSC6-T7. HEK 293T cells were seeded into 35 mm wells to reach ~50-70% confluency when transfected. Confluency is a term commonly used as an estimate of the number of adherent cells in a dish or flask, referring to the proportion of the surface covered by a monolayer of cells. Four hours before transfection, replace the medium with 3 ml DMEM containing 10% FCS. All recombinant plasmids were prepared according to the QIAGEN plasmid preparation kit. Ca for DNA 2+ The kit for phosphate co-precipitation was from Invitrogen.
[0123] Cells were co-transfected with the plasmids at the following final concentrations:
[0124]
[0125] Add the mentioned plasmids diluted in HO to the 2 Add the mixture to another Eppendorf tube containing HEPES buffer with shaking and incubate a...
Embodiment 3
[0133] Example 3: Amplification of Rescued Mumps JL2 Virus
[0134] Rescued rMuV JL2 The P0 generation of FL virus was propagated in WHO-approved Vero cells. Vero cells were maintained as a monolayer in DMEM supplemented with 5% FBS, and when confluency reached 70-80%, rescued rMuV had been passaged using 0.5-1.0 mL of rescued virus in a total volume of 5.0 mL of OPTIMEM. JL2 The P0 passage of FL virus infects Vero cells. Incubate the cells at 37 °C and 5% CO 2 Incubate for 1 hour. After incubation, the inoculum was replaced with DMEM+5% FBS. Infected cells were split after 2 days, and 90-100% syncytia formation was observed after 5 days of infection (5-dpi). Cell-free (CF) and cell-associated (CA) fractions have been collected to obtain laboratory-scale virus stocks up to 1 × 10 in the collected cell-free fraction (CF). 4 pfu / mL to 1×10 in the collected cell-associated fraction (CA) 6 Titers in pfu / mL.
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