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Preparation method and application of EV71 type full length infectious clone with luciferase tag

An infectious cloning, EV71 technology, applied in the biological field, can solve problems such as lack of treatment methods

Inactive Publication Date: 2012-07-11
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is still a lack of effective treatments

Method used

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  • Preparation method and application of EV71 type full length infectious clone with luciferase tag
  • Preparation method and application of EV71 type full length infectious clone with luciferase tag
  • Preparation method and application of EV71 type full length infectious clone with luciferase tag

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1: a kind of preparation method of the EV71 type full-length infectious clone with Rluc label, its steps are:

[0085] (1) Extraction of total RNA:

[0086] Get 140ul of EV71 virus liquid (mentioned above), extract the total RNA of EV71 according to the requirements of the QIAamp Viral RNA (purchased from Qiagen company) kit instructions, use Thermo Scientific NanoDrop 2000 (purchased from Thermo company) to measure the concentration of RNA, The quality of the RNA was detected by agarose gel electrophoresis at a concentration of 1% (w / v), and stored at -80°C for future use.

[0087] (2) RT-PCR amplification of EV71:

[0088]The whole genome of EV71 was divided into four parts to design primers (these four parts were named as F1, F2, F3 and F4 respectively). Using the total RNA of EV71 in the above step (1) as a template, DNA fragments F1, F2, F3 and F4 were respectively obtained using RT-PCR kit (purchased from Takara Company). The sequences of fragments F...

Embodiment 2

[0128] Embodiment 2: the ability of the EV71 full-length infectious clone with Rluc label to prepare EV71 virus, its steps are:

[0129] (1) Linearization of the plasmid: take 10 μg of the plasmid pACYC177-EV71-Rluc-FL, digest it with XbaI (described above), then add 100 μl of saturated phenol (purchased from Sinopharm Chemical Reagent Company) to the digested product, mix Evenly, centrifuge at 17000g for 5min, draw the upper layer liquid into a new centrifuge tube, add 100μl sterile water to the original centrifuge tube and mix, centrifuge at 17000g for 5min; absorb the upper layer liquid and combine with the liquid obtained in the previous step (the total volume is about 200μl), add 200 μl of chloroform (purchased from Sinopharm Chemical Reagent Company) was mixed, and centrifuged at 17000g for 5 min; the upper layer was taken into a sterile imported centrifuge tube (about 100 μl), and 10 μl of pH5.2, 3mol / l sodium acetate (purchased from Sinopharm Group Chemical Reagent Com...

Embodiment 3

[0132] Example 3: The EV71 full-length infectious clone with the Rluc tag has the ability to produce EV71 stably carrying the Rluc gene, and the steps are:

[0133] Inoculate 1.5 × 10 in a 12-well cell culture plate 5 Vero cells, when the confluence reached 60-70%, were infected with the P0 generation virus at 2, 6, 14, 16, 18, 20, 22 and 24 hours after the RNA transfected with pACYC177-EV71-Rluc-FL entered the Vero cells Vero cells, 37°C, 5% (v / v) CO 2 After culturing in the incubator for 24 hours, add 1ml of PBS to the wells, discard the PBS in the wells, add 200μl of cell lysate to treat the cells, mix the cell lysates in the wells, and take 20μl into a white 96-well plate , and 100 μl of substrate was added, and the activity of Rluc was detected using a multifunctional plate reader according to the requirements of the Luciferase Assay System instruction manual ( Figure 4 ). The activity of Rluc shows that the P0 generation virus produced by the full-length infectious c...

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Abstract

The invention discloses a preparation method and application of EV71 full length infectious clone with a luciferase tag, belonging to the field of biotechnology. The preparation method comprises the steps of: (1) extracting total RNA; (2) amplifying EV71 through RT-PCR; (3) constructing an EV71 subclone; (4) constructing a recombinant clone containing an EV71 gene; and (5) constructing an EV 71 full length infectious clone with a Rluc tag. Experiments of cytopathic effect, Rluc activity detection, virus plaque, drug inhibition and the like show that the EV71 full length infectious clone with a tag is successfully obtained. The invention has wide application values in the aspects of animal models, virus replication and pathogenesis, drug screening and mechanism of drug action, development of vaccines and diagnostic reagents and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for preparing a full-length infectious clone of enterovirus 71 with a Rluc label, and also relates to a method for preparing a virus with an EV71 full-length infectious clone with a Rluc label capabilities and applications in drug screening. Background technique [0002] Enterovirus 71 (EV71) belongs to the Enterovirus genus of the family Picornaviridae. Its genome is a single-stranded positive-sense RNA with a length of about 7.5 kb and a code of about 2200 amino acids. The polyprotein encoded by the genome can be cleaved into three precursor proteins (P1, P2, P3). Among them, the P1 precursor protein can cleave four viral structural proteins (VP1, VP2, VP3 and VP4). The P2 and P3 precursor proteins can be cleaved into seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D); the P3 region is highly conserved during evolution. The two sides of the ORF ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N7/01C12Q1/70C12Q1/68C12R1/93
Inventor 张波史佩勇许文波袁志明邓成林商宝娣贾凡
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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