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Construction method and application of Israeli acute paralysis virus (IAPV) infectious clone

A technology of infectious cloning, IAPV-BJ2, applied in the biological field to achieve obvious sensitivity and high efficiency

Active Publication Date: 2020-02-11
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, full-length infectious clones of many RNA viruses have been reported, including many insect RNA viruses, but no infectious clones of bee Israel acute paralysis virus have been reported.

Method used

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  • Construction method and application of Israeli acute paralysis virus (IAPV) infectious clone
  • Construction method and application of Israeli acute paralysis virus (IAPV) infectious clone
  • Construction method and application of Israeli acute paralysis virus (IAPV) infectious clone

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, the construction of honeybee IAPV virus infectious clone

[0035] The construction method of honeybee IAPV virus infectious clone, its steps are:

[0036] 1. Segmented synthesis of IAPV-BJ2 strain genome sequence:

[0037] Construction of cDNA infectious clones with full-length IAPV genome: select IAPV-BJ2 strain sequence, GenBank: MG599488.1; divide into three fragments for gene synthesis, and these three fragments are named fragment 1, fragment 2 and fragment 3 respectively , wherein Fragment 1 is T7+ from GenBank: MG599488.1 5' end 1-3823 nucleotide sequence (T7 promoter sequence connected to its 3' end from GenBank: MG599488.1 5' end 1-3823 A nucleotide fragment composed of nucleotides), fragment 2 is the 3804-6873 nucleotide sequence from the 5' end of GenBank: MG599488.1 (from the 3804-6873 nucleotide sequence at the 5' end of GenBank: MG599488.1 nucleotide fragment), and fragment 3 is a fragment obtained by joining the end of the 5' end 6855-9599 ...

Embodiment 2

[0041] Example 2, IAPV virus infectious clone successfully rescued the virus with the same growth trend as the wild-type virus

[0042] 1. PCR amplification and purification of viral genome: Use pACYC-IAPV plasmid as a template and use high-fidelity enzymes for PCR amplification. The reaction system is 2×buffer MIX: 25ul, water: 19ul, primer (5'-TAATACGACTCACTATAGGGACTTTTATGTCCCTACGTACAATTTTCGCCGAAATT-3 '(SEQ ID NO: 8)) 1 ul, primer (5'-TTTTTTTTTTTTTTTTTAAATTTACCTAATTCGAAAATTTTG-3' (SEQ ID NO: 9)) 1 ul, total system 50 ul. The amplification conditions are: 98°C for 30s, 98°C for 8s, 63°C for 20s, 72°C for 6min, 72°C for 10min, 4°C for 10min, 32 cycles. After the reaction, add 1ul dpnI enzyme (purchased from Beijing Quantum Shikini Co., Ltd.) at 37°C for 1 hour. Then, the PCR product was purified using a PCR product purification kit (purchased from Kangwei Century Co., Ltd.), and its concentration was measured.

[0043] 2. In vitro transcription of RNA: take the DNA purified ...

Embodiment 3

[0046] Embodiment 3, the application of IAPV virus infectious clone in antiviral drug screening, its steps are:

[0047] 1. Inhibitory effect of ginsenosides on honey bee virus

[0048] Take the newly emerged honeybees, put 30 honeybees into each cage, and raise them indoors for 24 hours (temperature 33°C, humidity: 50%), and feed them sugar water with a mass volume fraction of 50%. After 24 hours, take out the dead bees, and catch the bees so that the number of bees in the cage is 25. Then, put the bees in a refrigerator at 4°C for 15 minutes, then take the bees out and put them on ice, and then inject them into the backboard, and wash the needles with DEPC-treated water after each injection. A total of 4 groups, 25 animals in each group, each injection volume is 4.5ug, 4 groups are: 1. Blank group, 2. Injection of PBS, feeding sugar water group, 3. Injection of IAPV, feeding sugar water group, 4. Injection IAPV, fed 100ug / ml ginsenoside (purchased from Beijing Jingzhe Yong...

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Abstract

The invention discloses a construction method and application of an Israeli acute paralysis virus (IAPV) infectious clone. The IAPV infectious clone is prepared from the steps that (1) segmented synthesis of the IAPV BJ2 strain gene sequence is conducted; and (2) the IAPV infectious clone is assembled and constructed. Through experiments of a fluorescent quantitative test, a western blotting test,a toxicity experiment, drug inhibition, poison applying of living bees and the like, it is proven that the constructed IAPV infectious clone can rescue viruses with same symptoms as IAPV wild virus infected bees, and has the wide application value in the aspects of animal models, virus replication and pathogenesis, drug screening and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an IAPV infectious clone, a preparation method of the IAPV infectious clone, and the application of a reporter virus rescued by the clone in antiviral drug screening. Background technique [0002] Bee Israel Acute Paralysis Virus (IAPV) was isolated and identified in Israel in 2007. The virus can infect individual bees at various stages of development (eggs, larvae, pupae, adult bees) and bee colonies at all levels (worker bees, drones and queen bees). Since the virus was discovered in Israel, it has been found to exist widely all over the world. In recent years, it has also been found to be prevalent in Asia, especially in my country, where bee colonies are more common in winter. In some areas, its bee colony infection rate is as high as 96%. Its typical morbidity symptom is: sick honey bee body color darkens, fluff comes off, and with wing tremor, paralysis convulsion d...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/86C12N15/66C12N1/21A61K49/00C12R1/19
CPCA61K49/0008C12N15/66C12N15/86C12N2770/22043
Inventor 侯春生邓帅杨卅代平礼吴艳艳王强刁青云
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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