Attenuated virus of flavivirus and application of attenuated virus

A yellow fever virus, virus technology, applied in the direction of virus, antiviral agent, inactivation/attenuation, etc.

Active Publication Date: 2021-07-30
湖北彰骏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are no effective antiviral drugs and specific treatment methods against flavivirus infection, and there are only 3 kinds of flavivirus vaccines for human prevention. Flavivirus infection is of strategic importance

Method used

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  • Attenuated virus of flavivirus and application of attenuated virus
  • Attenuated virus of flavivirus and application of attenuated virus
  • Attenuated virus of flavivirus and application of attenuated virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1: Construction and virus rescue of flavivirus attenuated strain clone

[0115] 1. Cloning construction of flavivirus attenuated strain

[0116] Schematic diagram of the cloning construction of attenuated flavivirus strains figure 1 shown.

[0117] Using the plasmid DNA of the infectious clone of the wild-type (WT) West Nile virus (WNV) strain as the backbone, the sequence from SLI to CS2 (including the 5' Terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3 ), dumbbell region 1 (DB1), repeat circularizing sequence region 2 (RCS2), dumbbell region (DB2), circularizing sequence region 2 (CS2)) deletion, retaining circularizing sequence region 1 (CS1) in the 3' untranslated region ) and the short hairpin-3' stem-loop region (sHP-3'SL) after circularizing sequence region 1, and inserting a poly(A) sequence...

Embodiment 2

[0132] Construction and virus rescue of other West Nile virus attenuated strain clones of embodiment 2

[0133] 1. Construction of clones of other West Nile virus attenuated strains

[0134] Schematic diagram of the clone construction of other West Nile virus attenuated strains as shown in figure 2 shown.

[0135] Using the plasmid DNA of the infectious clone of the West Nile virus (WNV) strain of the wild type (WT) in Example 1 as a backbone, the sequence from SLI to CS3 in the 3' untranslated region (3'UTR) of WNV (including SLI, SLII, RCS3, SLIII, SLIV, CS3) deleted, retaining dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region 2 (DB2), circularization sequence in the 3' untranslated region Region 2 (CS2), Circularization Sequence 1 (CS1), and the short hairpin-3' stem-loop region after cyclization Sequence 1 (sHP-3'SL), with deletion of the sequence from SLI to CS3 The poly(A) sequence (130 adenosine nucleotides) was inserted into ...

Embodiment 3

[0144] Embodiment 3: Flavivirus attenuated strain and wild-type virus growth curve comparison

[0145] 1. Virus Titer Determination

[0146] The titer of the virus was determined by the plaque method, and the specific steps were as follows:

[0147] Inoculate 1×10 cells in each well of a 24-well cell culture plate 5 For each BHK-21 cell, when the cell confluency reaches 90%, discard the medium in the well, and add 100 μl of the poly of the attenuated flavivirus collected in Step 3 of Example 1 diluted 10 times with DMEM medium containing 2% FBS. (A) P0 generation virus, adsorb for 1 hour at 37°C, and shake fully every 15 minutes. After the adsorption is complete, the virus liquid in each well is discarded, and the DMEM medium containing 2% FBS and the cover of 2% methylcellulose are added, and the virus solution is incubated at 37°C and 5% CO. 2 Cultured in an incubator for 3 days, stained with staining solution containing 1% crystal violet and 3.7% formaldehyde after plaqu...

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Abstract

The present invention relates to an attenuated virus of a flavivirus and application of the attenuated virus. The attenuated virus comprises a polyadenylic acid (poly(A)) sequence, wherein the poly(A) is used for replacing a part of nucleotide sequence of a 3' untranslated region (3' UTR) of the yellow disease virus, so that the 3' untranslated region (3' UTR) of the attenuated yellow disease virus obtained after the part of nucleotide sequence of the yellow disease virus is replaced at least retains a 3'-end stem-loop region (3' SL). The attenuated virus has growth characteristics similar to those of a wild type virus, and is high in titer and good in hereditary stability. Moreover, the attenuation is fully attenuated, the safety is high, the generation of high-level neutralizing antibody titer can be induced by single immunization, the attack of high-dose wild-type viruses can be resisted, complete immune protection can be provided, and the attenuated vaccine strain can be used for preparing safe and effective attenuated vaccine strains.

Description

technical field [0001] The disclosure belongs to the field of biotechnology, and in particular relates to an attenuated virus of the genus Flavivirus and its use. Background technique [0002] The Flavivirus genus belongs to the Flaviviridae family and includes more than 70 viruses, most of which are transmitted by mosquitoes or ticks. Among them, Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVEV), dengue virus (DENV) and Zika virus (ZIKV) are the most Important human pathogen with high morbidity and mortality. Severe flavivirus infection disease including hemorrhagic fever (YFV and DENV), encephalitis and neurological sequelae (JEV, TBEV, WNV, SLEV and MVEV), can lead to death, among which the mortality caused by JEV, YFV, TBEV and DENV can up to 5% to 30%. [0003] At present, there are no effective antiviral drugs and spec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/04C12N5/10A61K39/12A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2770/24121C12N2770/24152C12N2770/24162C12N2770/24134A61K2039/5254Y02A50/30
Inventor 张波叶寒青张亚男李娜张秋艳邓成林袁绍鹏占顺利高磊
Owner 湖北彰骏生物科技有限公司
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