Preparation of recombinant slow virus of shRNA (Short Hairpin Ribonucleic Acid) of target GPMV NP and M genes

A recombinant lentivirus and targeting technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problem of low transgenic efficiency and achieve wide application and far-reaching research effects

Inactive Publication Date: 2012-08-01
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology helps create better ways to protect birds from harmful bacterial or virus attacks while still giving them their healthy flocks.

Problems solved by technology

This patents discusses how different types of pathogen called Goa pseudoridaviruses causing various human illnesses such as measles fever syndrome, porcinosis, encephalitis, necropsia, tissue damage due to starfish import from China during spring outbreaks. Current treatments involve chemical agents like amantadienolides but these treatment options provide limited effectiveness against other etiotropic modes of transmission. There also exist gene therapy techniques targeting certain proteins involved in protecting birds from disease challenges.

Method used

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  • Preparation of recombinant slow virus of shRNA (Short Hairpin Ribonucleic Acid) of target GPMV NP and M genes
  • Preparation of recombinant slow virus of shRNA (Short Hairpin Ribonucleic Acid) of target GPMV NP and M genes
  • Preparation of recombinant slow virus of shRNA (Short Hairpin Ribonucleic Acid) of target GPMV NP and M genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Design of RNAi target and construction of shRNA lentiviral vector

[0027] Using Ambion's siRNA online design software and design principles, screen the NP and M genes of the GPMV NA-1 strain for RNAi targets, and at the same time perform BLAST analysis on the obtained target sequences in GenBank; target siRNA oligonucleotides The sequence was sent to Shanghai Sangong for synthesis, and the synthesized oligonucleotide sequence was annealed to form a double strand, which was ligated with the linearized pLKD-EGFP vector after double digestion with endonuclease AgeⅠ and EcoRI, transformed into competent Escherichia coli DH5α, and picked for recombinant Positive clones were identified by PCR and sequencing (sequence analysis by Shanghai Sangon Company), and pLKD-shNP and pLKD-shM lentiviral vectors were produced.

[0028] The primer sequence of positive clones identified by PCR: F: 5'-AATGGACTATCATATGCTTACCGTAACTTG-3',

[0029] R: 5'-TGTCTGTTGCTATTATGTCTACTATTC...

Embodiment 2

[0030] Example 2 lentiviral packaging the

[0031] 24 hours before transfection, the 293T cells in the logarithmic growth phase were digested with 0.25% trypsin, and the cells were counted, and the cell density was adjusted to 5×10 6 cells / ml, reseeded in T75 cell culture flasks, and transfected when the cell density reached 70%-80%. Add the DNA solution of the plasmid in the lentiviral packaging system pLKD-shNP or pLKD-shM: 9μg; pCMV-dR8.2: 6.75μg and pCMV-VSV-G: 2.25μg to 500μl double antibody-free, serum-free DMEM culture medium and 54μl, mix well, and incubate at room temperature for 20min. Transfer the mixture of DNA and FuGENG HD transfection reagent to the culture medium of 293T cells and mix them evenly, culture them in a cell incubator at 37°C and 5% CO2, and discard the medium containing the transfection mixture after culturing for 6 hours. 12 ml of cell culture medium containing 10% FBS serum was added to the culture dish, and culture was continued for 48 hours...

Embodiment 3

[0032] Example 3 Determination of lentivirus titer by LaSRT method

[0033] 293T cells with 2×10 4 Each well was inoculated in a 96-well culture plate, and after 12 hours, 8 wells were used as a group, and replaced with DMEM culture solution containing 8 μg / ml Polybrene and 10% FBS, 90 μl / well. Add 10 μl of the lentivirus stock solution to be tested into the first well, and then sequentially dilute each well 10:1 to set up and measure 3 groups of duplicate wells. After 48 hours of infection, observe the changes in the number of GFP-positive cells in each well under a fluorescent inverted microscope in the direction of concentration from high to low, determine the metering well (well n), and count the number of positive cells m, virus titer (TU / ml)=m×10 n+1 .

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Abstract

The invention discloses a method for mediating RNAi (RNA interfere) by utilizing slow-virus vectors. The method comprises the following detailed steps of: firstly carrying out homologous analysis on the nucleic acid sequence of goose paramyxovirus NA-1 NP and M genes, finding conserved regions, and designing out siRNA on the conserved regions; cloning the siRNA to slow-virus expression plasmid pLKD-EGFP, then commonly transfecting the slow-virus expression plasmid pLKD-EGFP with pCMV-dR8.2 and pCMV-VSV-G in 293T cells, collecting transfected cell supernatant, forming high-titer slow-virus vectors expressing the siRNA by ultracentrifugation, purification and concentration, then carrying out transduction on the high-titer slow-virus vectors into target cells, and enabling the slow-virus vectors to be stably expressed in the cells and exert the antiviral effect. The method disclosed by the invention has the advantages that the antiviral effect of the siRNA in a body can be really evaluated due to high-efficiency transduction rate and strict control system, the basis of the molecular biology can be laid for disease-resistance breeding of poultry animal molecules, and simultaneously, a new path is provided for preventing and treating the animal-based virus diseases such as newcastle diseases.

Description

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Claims

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Application Information

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Owner JILIN UNIV
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