Composition for preparation of low-toxicity rift valley fever virus, preparation method of composition and RVFV attenuated vaccine
A technology for Rift Valley fever virus and composition, applied in the fields of VFV attenuated vaccine, Rift Valley fever virus composition and preparation, can solve problems such as virulence recovery, reduce virulence, retain originality, and improve cell resistance. receptive effect
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Embodiment 1
[0074] This embodiment provides the construction method of RVFV whole genome cDNA recombination transcription plasmid, this method specifically comprises the following steps:
[0075] (1) Preparation of the whole genome cDNA fragment of RVFV:
[0076] The RVFV BJ01 virus strain is preserved in the Microorganisms & Viruses Culture Collection Center, Wuhan Institute of Virology, Chinese Academy of Sciences (MVCCC, WIV, CAS). RVFV virion diagram reference figure 1 shown. Viral genomic RNA in the cell supernatant was extracted using a viral RNA purification kit. After measuring concentration, utilize one-step method RT-PCR kit to carry out RT-PCR with virus genome RNA as template, utilize RT-PCR to amplify respectively RVFV-L, three cDNA gene fragments of RVFV-M and RVFV-S (primers refer to shown in Table 1). In Table 1, the forward primer of RVFV-L is L-T7-for, and the reverse primer is L-T7-rev, corresponding to SEQ ID NO.8 and SEQ ID NO.9 respectively; the forward primer of...
Embodiment 2
[0091] This example provides a method for obtaining the recombinant virus rRVFV. Specifically include the following steps:
[0092] BSR-T7 cells in good growth state were seeded in six-well plates (4 × 10 5 cells / well), when the density was 70%-80% when grown to monolayer, pVSV-RVFV-L-T7, pVSV-RVFV-M-T7 and pVSV- 1 μg of each of the three plasmids of RVFV-S-T7 was co-transfected into BSR-T7 (see the kit instructions for specific operations), and placed in 5% CO 2 , Cultivate in a 37°C incubator for 8h to 12h. After the supernatant was discarded, DMEM medium containing 2% FBS was added to continue culturing for 3d-5d. Observe under the microscope every day, and collect the supernatant when the cells appear lesions (Cytopathic effect, CPE). A part of the supernatant was transferred to BHK-21 cells and amplified as the P1 generation virus; the other part of the supernatant was stored as a seed virus at -80°C (P0 generation virus). The collected cells were used for subsequent...
Embodiment 3
[0094] This example provides a NSs gene modification method for the S segment of a recombinant virus. Specifically include:
[0095] Design primers S-eGFP-F and S-eGFP-R (shown in the primer table with reference to Table 1), primers S-eGFP-F and S-eGFP-R are the seventh primer and the eighth primer, and the primer sequence is as SEQ ID As shown in NO.14 and SEQ ID NO.15, eGFP with a homologous sequence to the plasmid vector was amplified by PCR; similarly, SUTR-F and SUTR-R were used as primers, and the pVSV-RVFV-S-T7 plasmid was used as a template to amplify The third gene fragment pVSV-RVFV-SUTR containing the encoding NP protein was added (the primers are shown in the primer table of Table 1). The primers SUTR-F and SUTR-R are the ninth primer and the tenth primer of the present invention, and the primer sequences are shown in SEQ ID NO.16 and SEQ ID NO.17 respectively. Both ends of eGFP (the fourth gene segment) and pVSV-RVFV-SUTR (the sixth gene segment) contain homolog...
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