Attenuated JEV (Japanese encephalitis virus) having enhanced replicability, and preparation method and application of attenuated JEV (Japanese encephalitis virus) having enhanced replicability

A technology of replicative and infectious cloning, applied in the field of biomedicine, can solve the problems such as the lack of research on RNA elements and the unclear function of RNA elements, so as to speed up the process of cultivation and virus pathogenic mechanism research, reduce pathogenicity, The effect of increasing production

Active Publication Date: 2020-06-26
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

[0004] The RNA structure of the non-coding region in the genomic RNA of the flavivirus and the NS5 protein have a very important impact on the survival and spread of the virus, although the non-coding region of the flavivirus is currently The research on regional RNA has been carried out in depth, but the function of the RNA element is still unclear, and the research on the RNA element SL-IV is still less

Method used

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  • Attenuated JEV (Japanese encephalitis virus) having enhanced replicability, and preparation method and application of attenuated JEV (Japanese encephalitis virus) having enhanced replicability
  • Attenuated JEV (Japanese encephalitis virus) having enhanced replicability, and preparation method and application of attenuated JEV (Japanese encephalitis virus) having enhanced replicability
  • Attenuated JEV (Japanese encephalitis virus) having enhanced replicability, and preparation method and application of attenuated JEV (Japanese encephalitis virus) having enhanced replicability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of pACYC-SA14-M-NS5-3'UTR infectious clone

[0044] 1. Using the plasmid pACYC-JEV-SA14 / U14163 (abbreviated as pACYC-SA14 / U14163, the same below) as a template and pACYC-full4 and WH-HDVr-F as primers, carry out PCR reaction. The PCR reaction system is: 5×Q5 Reaction Buffer 10 μL, 2.5M dNTP mix 4 μL, pACYC-full4 2.5 μL (10 μmol / μL), WH-HDVr-F 2.5 μL (10 μmol / μL), Q5 High-Fidelity DNA Polymerase 0.5 μL, template 0.5 μL, make up to 50 μL with deionized water. The PCR reaction conditions were: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 20 s, and 30 cycles, and finally extension at 72°C for 5 min. The resulting product was named HDVr-SV40 poly(A).

[0045] 2. Combine fragment HDVr-SV40 poly(A) (278bp) and M-NS5-3'UTR (1 884bp) in equal moles (the total mass of the two fragments is 700ng, HDVr-SV40 poly(A) 90ng, M-NS5 -3'UTR 610ng) was assembled by fusion PCR reaction, there wa...

Embodiment 2

[0050]Example 2 Construction of pACYC-SA14-M-NS5 infectious clone

[0051] 1. Use the plasmid pACYC-SA14-M-NS5-3'UTR as a template, and XbaI-F and NS5-R as primers for PCR amplification. The PCR reaction system refers to formula example 1. The PCR reaction conditions were: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 30 cycles, and finally extension at 72°C for 5 min. The resulting product was named M-NS5 (1 305bp).

[0052] 2. Using the plasmid pACYC-SA14 / U14163 as a template and 3'UTR-F and pACYC-full4 as primers, perform PCR amplification. The PCR reaction system refers to formula example 1. The PCR reaction conditions were: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 25 s, and 30 cycles, and finally extension at 72°C for 5 min. The resulting product was named SA14-3'UTR (816bp).

[0053] 3. The fragment M-NS5 (1 305bp) ...

Embodiment 3

[0055] Example 3 Construction of pACYC-SA14-M-3'UTR infectious clone

[0056] 1. Using the plasmid pACYC-SA14 / U14163 as a template and XbaI-F and NS5-R as primers, carry out PCR amplification (see Example 1). The PCR reaction system refers to formula example 1. The PCR reaction conditions were: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 25 s, and 30 cycles, and finally extension at 72°C for 5 min. The resulting product was named SA14-NS5 (1305bp).

[0057] 2. Using the plasmid pACYC-SA14-M-NS5-3'UTR as a template, 3'UTR-F, and pACYC-full4 as primers, perform PCR amplification. The PCR reaction system refers to formula example 1. The PCR reaction conditions were: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 30 cycles, and finally extension at 72°C for 5 min. The resulting product is named M-3'UTR (817bp)

[0058] 3. The fra...

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Abstract

The invention discloses an attenuated JEV (Japanese encephalitis virus) having enhanced replicability, and a preparation method and application of the attenuated JEV (Japanese encephalitis virus) having enhanced replicability, and belongs to the technical field of biomedical science. The attenuated JEV disclosed by the invention is a Japanese encephalitis virus obtained in a manner that the 644th-site aspartic acid of an NS5 protein of the Japanese encephalitis virus is mutated into threonine, the 248th-site base T in a 3' noncoding region is mutated into C, the 254th-site base A is mutated into G and the 258th-site base A is mutated into G. A mutation strain obtained by the method disclosed by the invention has obviously-improved reproduction capacity, and besides, the pathogenicity of the mutation strain is reduced, so that when the mutation strain is used for vaccine production, the yield can be increased, and the cost can be reduced. The time period for obtaining the mutation strain by the method disclosed by the invention is short, operations are simple and convenient, and a process for incubating attenuated strains and researching a pathogenic mechanism of viruses can be accelerated.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an attenuated JEV with enhanced replicability and its preparation method and application. Background technique [0002] Japanese encephalitis (JE) (referred to as JE) is an insect-borne zoonotic disease caused by Japanese encephalitis virus (JEV). Pigs are usually used as storage hosts before JEV infects humans. After being infected with the virus, pregnant sows will suffer from miscarriage, stillbirth, fetal malformation and mummified fetuses; boars will suffer from orchitis and reproductive disorders, which will have a huge impact on the animal husbandry industry. At the same time, it also poses a huge threat to human health and is one of the most common insect-borne diseases of the human central nervous system. [0003] JEV is a single-stranded positive-strand RNA virus coated with an envelope, with a size of 11Kb. The genome has a long open reading frame (ORF...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/40A61K39/12A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2770/24121C12N2770/24122C12N2770/24134Y02A50/30
Inventor 亓文宝刘乐乐张又月廖明邢金超梁佳琪
Owner SOUTH CHINA AGRI UNIV
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