A kind of construction method and application of iapv infectious clone

A technology of infectious cloning and construction method, applied in the application of reporter virus in the screening of antiviral drugs, and the preparation of IAPV infectious clones, to achieve the effects of shortening the research and development cycle, wide application value, obvious sensitivity and high efficiency

Active Publication Date: 2021-09-21
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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  • A kind of construction method and application of iapv infectious clone
  • A kind of construction method and application of iapv infectious clone
  • A kind of construction method and application of iapv infectious clone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1, bees build IAPV viral infectious clones Example

[0035] The method of constructing bees IAPV viral infectious clone, which is the step of:

[0036] 1, IAPV-BJ2 strain genomic sequence of the synthetic fragment:

[0037] Construction of cDNA infectious clone of the full-length genome having IAPV: Select IAPV-BJ2 strain sequence, GenBank: MG599488.1; gene synthesis fragments were divided into three, these three fragments were named fragment 1, fragment 2 and fragment 3 wherein the fragment of T7 + 1 from GenBank: MG599488.1 5 'end of the nucleotide sequence 1-3823 (T7 promoter sequence and its 3' end of the connection from GenBank: MG599488.1 5 'end of the first 1-3823 nucleotide fragment consisting of nucleotides), fragment 2 was from GenBank: MG599488.1 5 'end of the nucleotide sequence 3804-6873 (from GenBank: MG599488.1 5' end of the first position 3804-6873 nucleotide fragments), fragment 3 from GenBank: 5 MG599488.1 'end of the fragment position 6855-9599 (from GenB...

Embodiment 2

[0041] Example 2, IAPV viral infectious clones successfully rescued wild type virus with the same growth trend virus

[0042] . 1, the viral genome PCR Amplification and Purification: The plasmid pACYC-IAPV as a template, PCR amplification was performed using the high fidelity enzyme, the reaction system was 2 × buffer MIX: 25ul, water: 19 uL, primer (5'-TAATACGACTCACTATAGGGACTTTTATGTCCCTACGTACAATTTTCGCCGAAATT-3 '(SEQ ID 8)) 1ul, primer (5'-TTTTTTTTTTTTTTTTTAAATTTACCTAATTCGAAAATTTTG-3' (sequence 9)) 1ul, total system 50ul. Amplification conditions were: 98 ℃ 30s, 98 ℃ 8s, 63 ℃ 20s, 72 ℃ 6min, 72 ℃ 10min, 4 ℃ 10min, 32 cycles after the completion of the reaction, to each tube was added 1ul dpnI enzyme (purchased from Beijing whole. formula CICC), the reaction 37 ℃ 1 hour. Then using PCR Purification Kit (commercially available from Kang Century Company) The PCR product was purified, and the measured concentration.

[0043] 2, in vitro transcribed RNA: Take further purified on a DNA...

Embodiment 3

[0046] Example 3, application in IAPV viral infectious clone antiviral drug screening, the steps of:

[0047] First, ginseng saponin inhibition of bee viruses

[0048] Take the new room bee, bee placed in a 30 per cage, reared 24h (temperature 33 ℃, humidity: 50%), fed volume fraction of 50% by mass of sugar. After 24h, remove dead bees, bees and catch the number of bees are 25 cages. Then, first the bees into the refrigerator at 4 ℃ frozen 15min, then taken out on ice bees, infection and injection manner through the backplane, each with DEPC treated water after the injection needle wash. 4 groups of 25, in an amount of 4.5 ug per injection, four groups were: 1. control group, PBS injection 2, feeding glucose group, 3 injections IAPV, feeding sugar group, 4 injections. IAPV, feeding ginsenosides 100ug / ml of (purchased from Beijing Jing Zhe Yongxing biotech companies) group. Two parallel experiments each done, normal feeding after injection (temperature 33 ℃, humidity: 50), daily...

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Abstract

The invention discloses a construction method and application of an IAPV infectious clone. An IAPV infectious clone, the preparation steps of which are: (1) segmental synthesis of the IAPV virus BJ2 strain gene sequence; (2) assembly and construction of the IAPV virus infectious clone. The present invention proves through fluorescence quantitative test, western blotting test, toxicological test, drug inhibition, live bee poison feeding and other experiments that the IAPV virus infectious clone constructed by the present invention can rescue bees with the same symptoms as the IAPV wild-type virus infected bees. Viruses, and has a wide range of application value in animal models, virus replication and pathogenic mechanism, drug screening, etc.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and more particularly to IAPV infectious clones, and also relates to the preparation method of IAPV infectious clones, and the application of the reported virus in anti-viral drugs in the anti-viral drug screening. Background technique [0002] Bee Israel Acute Paralyvirus (IAPV) was separated from Israel in 2007. The virus can infect individual (eggs, larvae, pupa, bee) and bee colony, bee, and bee and royal king). After discovery this virus, it has found extensive existence in all parts of the world. In recent years, it has been popular in Asia, especially in the winter of my country, and has become one of the most serious diseases that endanger my country's winter hive. Some areas have a hive infection rate up to 96%. Its typical morbidity is: sick honey bee body color darkening, fluff off, accompanied by a winged tremble, gradually paralyzed twitching. The condition is similar to ABPV, and its biologi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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