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Visible recombinant virus of human enterovirus 71 and application thereof

A technology of enterovirus and recombinant virus, which is applied in application, virus/bacteriophage, recombinant DNA technology, etc., can solve the problems of inability to image viruses in real time, and achieve good application prospects, stable genes, and simple operation

Inactive Publication Date: 2015-03-18
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the most commonly used method for fluorescently labeling proteins is the traditional immunofluorescence technique, but this technique cannot perform real-time imaging of viruses
The total length of the EV71 virus genome is only 7.4kb, 3C pro The length of the gene is about 500bp, and it is difficult to insert the coding gene of GFP, RFP or other luciferases with a length of about 1kb into the viral genome, so the visual reporter virus of EV71 has not been reported yet

Method used

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  • Visible recombinant virus of human enterovirus 71 and application thereof
  • Visible recombinant virus of human enterovirus 71 and application thereof
  • Visible recombinant virus of human enterovirus 71 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the construction of recombinant EV71 virus

[0042] The AH / 08 / 06 strain is a domestic isolate of enterovirus type 71, and the full-length cDNA corresponding to its genomic RNA is shown in sequence 3 of the sequence table (the 5387th to 5935th nucleotides from the 5' end are 3C pro Gene, 3C shown in sequence 5 of the coding sequence listing pro protein).

[0043] 1. Enterovirus 71 3C pro Construction and Identification of Recombinant Plasmids

[0044] 1. Construct the plasmid of full-length infectious cDNA clone A12 of EV71 AH / 08 / 06 strain

[0045] Synthesize the double-stranded DNA molecule shown in sequence 3 of the sequence table, and insert it between the SnaBI and MluI restriction sites of the pBR322 plasmid to obtain the recombinant plasmid A (also known as the full-length infectious cDNA clone carrying the EV71 AH / 08 / 06 strain A12 plasmid, also known as enterovirus 71 recombinant plasmid).

[0046] 2. Design and synthesize the following primers ...

Embodiment 2

[0076] Embodiment 2, indirect immunofluorescence analysis

[0077] 1. Preparation of antigen sheets

[0078] Use 8-well sterilized cell culture slides to culture RD cells. After the cells are 90% confluent, inoculate enterovirus 71, absorb at 37°C for 1 hour, discard the supernatant, and add DMEM maintenance medium containing 2% FBS , placed at 37°C 5% CO 2 incubator for cultivation. Samples were taken 6, 12, and 24 hours after virus inoculation, the cells were washed three times with PBS buffer, and pre-cooled acetone (500 μl / well) was added and fixed overnight at -20°C. After taking out the slides, dry them at room temperature and store them at -20°C.

[0079] 2. Indirect immunofluorescence analysis

[0080] Take out the antigen sheet prepared in step 1, equilibrate to room temperature, add 100 μl 1:200 times diluted monoclonal antibody specific to the VP1 protein of enterovirus 71, and incubate for 1 hour in a humid box at 37°C, and remove the antigen The slices were s...

Embodiment 3

[0082] Embodiment 3, western blot analysis

[0083] Inoculate RD cells into a 48-well plate. After the cells grow to a monolayer, enterovirus 71 is inoculated at 37°C with 5% CO 2 Incubate in the incubator for 6 hours, then wash the cells three times with PBS buffer, add 0.25% trypsin to digest the cells until round and shrink, discard the trypsin, resuspend the cells with PBS buffer, add protein loading buffer, and boil in water Samples were loaded after 10 minutes, followed by SDS-PAGE (5% stacking gel, 80V, 12% separating gel, 120V, total electrophoresis time 1h) and immunoblotting. In western blot, use 1:1000 dilution of specific monoclonal antibody against enterovirus 71 VP1 protein or 1:100 dilution of anti-3C proMonoclonal antibody, using alkaline phosphatase-labeled goat anti-mouse secondary antibody diluted 1:1000 times. A blank control (Neg) not inoculated with enterovirus 71 was set.

[0084] see photos Figure 4 , where Figure A is the use of 3C against enterov...

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Abstract

The invention discloses a visible recombinant virus of human enterovirus 71 and an application thereof. In the invention, firstly a double-stranded DNA molecule is protected, wherein the double-stranded DNA molecule is obtained by inserting a coding gene labeled by a CCPGCC label into a 3C protease coding area of genomic DNA of the human enterovirus 71. The CCPGCC label is a polypeptide fragment represented as the sequence I in the sequence table. An expression kit, a recombinant vector, a transgenic cell line or a recombinant bacterium are all belongs to a scope of protection in the invention. The invention also protects a recombinant virus of the human enterovirus 71, wherein the coding DNA of a genomic RNA of the recombinant virus is represented as the sequence 4 in the sequence table. The visible recombinant virus of the human enterovirus 71 has an excellent application prospect in fundamental research.

Description

technical field [0001] The invention relates to an enterovirus 71 visualized recombinant virus and an application thereof, in particular to an enterovirus 71 3C protein visualized recombinant virus and an application thereof. Background technique [0002] Enterovirus 71 (EV71) is an important member of the genus Enterovirus in the family Picornaviridae. EV71 infection mainly causes Hand-Foot-Mouth Disease (Hand-Foot-Mouse Disease), which can lead to severe neurological syndrome and is the main cause of severe and fatal cases of Hand-Foot-Mouth Disease. Currently, there are no effective vaccines and specific antiviral drugs against EV71 clinically. [0003] The genome of EV71 is a single-stranded positive-strand RNA molecule, containing a single reading frame, encoding 4 structural proteins (VP1 protein, VP2 protein, VP3 protein and VP4 protein) and 7 non-structural proteins (2A protein, 2B protein, 2C protein protein, 3A protein, 3B protein, 3C protein and 3D protein). Th...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N7/01C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12Q1/70G01N33/577G01N21/64
Inventor 秦成峰曹瑞源秦鄂德韩剑峰李晓峰
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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