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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

Inactive Publication Date: 2019-11-22
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional attenuated live vaccine preparation method in which attenuated strains can be obtained by successive passages in different cell lines for multiple times has the defect of virulence recovery.

Method used

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  • Composition for preparation of low-toxicity rift valley fever virus, preparation method of composition and RVFV attenuated vaccine
  • Composition for preparation of low-toxicity rift valley fever virus, preparation method of composition and RVFV attenuated vaccine
  • Composition for preparation of low-toxicity rift valley fever virus, preparation method of composition and RVFV attenuated vaccine

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a composition for preparation of a low-toxicity rift valley fever virus, a preparation method of the composition and an RVFV attenuated vaccine, and relates to the field of biotechnology. According to a reverse genetic system, introduction of excess basic groups is avoided, the primitiveness of a viral genome is kept to the greatest degree, and the reverse genetic system can stably express a low-toxicity rift valley fever virus strain in the later period, the defect of toxicity recovery is avoided, and the limitation is broken that by means of a traditional method, an attenuated strain can be obtained through continuous passage many times.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a composition and a preparation method for preparing attenuated Rift Valley fever virus, and an RVFV attenuated vaccine. Background technique [0002] Rift Valley fever is an acute, febrile zoonotic disease caused by Rift Valley fever virus (RVFV), which is mainly transmitted through mosquito bites or contact with infected animals. infectious disease. A variety of domestic animals such as goats, sheep, cattle, and camels can be infected with RVFV, which can cause abortion and stillbirth in pregnant female animals. Humans have symptoms such as fever, hepatitis, meningitis, and hemorrhagic syndrome after infection. [0003] RVFV belongs to the order Bunyavirales, Phenuiviridae, and Phlebovirus. The diameter of the virus particle is about 90nm-110nm, spherical, with a capsule, and it is a single-strand negative chain and segmented. Segment of RNA virus. [0004] Currently, there are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/86C12N15/64A61K39/12A61P31/14
CPCA61K39/12A61P31/14C12N7/00C12N15/64C12N15/86C12N2760/12234C12N2760/12262C12N2760/20243Y02A50/30
Inventor 彭珂李淑芬朱向涛管真琼张玉兰
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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