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Lymphatic choriomeningitis virus for expressing luciferase gene as well as construction method and application of lymphatic choriomeningitis virus

The technology of a luciferase gene and a construction method is applied in the field of lymphocoriochoriomeningitis virus and its construction, which can solve the problems of large side effects and limited therapeutic effect of ribavirin, and achieve the effect of wide application value.

Pending Publication Date: 2022-03-25
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the broad-spectrum antiviral drug ribavirin, there are no FDA-approved vaccines and drugs specific for arenaviruses in clinical practice. However, ribavirin has limited therapeutic effects and relatively large side effects.

Method used

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  • Lymphatic choriomeningitis virus for expressing luciferase gene as well as construction method and application of lymphatic choriomeningitis virus
  • Lymphatic choriomeningitis virus for expressing luciferase gene as well as construction method and application of lymphatic choriomeningitis virus
  • Lymphatic choriomeningitis virus for expressing luciferase gene as well as construction method and application of lymphatic choriomeningitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of lymphatic choriomeningitis virus expressing luciferase gene.

[0054] 1.1 Construction of infectious clones carrying luciferase gene.

[0055] Lymphatic choriomeningitis virus consists of a large segment L (GenBank No.AY847350.1) and a small segment S (GenBank No.AY847351.1). The L segment is 7228 nt in length, including the UTR regions at both ends and the IGR region in the middle. The L segment encodes Viral RNA-dependent RNA polymerase L and matrix protein Z. The S fragment is 3376nt in full length, encoding the nucleoprotein NP and envelope glycoprotein and envelope glycoprotein GPC of the virus. The cDNA sequences corresponding to the full-length L segment and S segment of the LCMV Armstrong strain were chemically synthesized through the DNA chemical synthesis service provided by a commercial company, including the UTR region at the 5' end and the 3' end and the IGR sequence in the middle.

[0056] After artificially synthesizing the tw...

Embodiment 2

[0064] Example 2: Identification of LCMV-Nluc recombinant virus.

[0065] The P1 generation virus to the P5 generation virus of the LCMV-Nluc in Example 1 were photographed respectively, and compared with the LCMV-WT virus, it was found that the two had no difference in morphological structure as figure 2 shown.

[0066] The P1-P5 recombinant virus was identified by PCR, and it was found that the Nluc gene existed stably, and there was no gene loss due to the influence of passage, such as image 3 shown.

[0067] LCMV-Nluc and LCMV-WT were amplified at the same MOI, and the two viruses were collected at different time points at 6h, 12h, 24h, 36h, 48h, and 72h to detect the expression of the NP gene. The expression level was used as a control, and it was found that the gene expression levels of the two were basically the same, see Figure 4 , indicating that the growth kinetics of the recombinant virus was consistent with that of the wild-type virus.

[0068] The virus wa...

Embodiment 3

[0070] Example 3: Application of LCMV-Nluc recombinant virus in drug screening at cell level in vitro.

[0071] Inhibitory effect of Ribavirin on LCMV-Nluc: Inoculate 20,000 / well Vero-E6 cells in each well of a 96-well cell culture plate at 37°C, 5% CO 2 Under culture conditions, when the confluence reaches 80-90%, add Ribavirin serially diluted with DMEM containing 2% fetal bovine serum (concentrations are respectively 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 , 3.90625μM), after incubation for 1h, add LCMV-Nluc virus solution to each well, the MOI is 0.1, 37℃, 5%CO 2 After 2 hours of adsorption in the incubator, the liquid in each well was discarded with a pipette tip, and Ribavirin serially diluted with DMEM containing 2% fetal bovine serum (concentrations were 1000, 500, 250, 125, 62.5, 31.25 , 15.625, 7.8125, 3.90625μM), 37°C, 5% CO 2 After culturing in the incubator for 48 hours, the supernatant in the wells was discarded, and 0.5ml PBS was added to the wells to...

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Abstract

The invention discloses a lymphatic choriomeningitis virus for expressing a luciferase gene as well as a construction method and application of the lymphatic choriomeningitis virus, and relates to the technical field of biology. The luciferase gene is inserted between the 5 'UTR of the LCMV virus genome and the N end of the NP protein sequence to construct the virus genome marked by the luciferase reporter gene, the result does not affect protein processing modification after virus translation, and the generated luciferase protein does not affect assembly of filial generation virus particles. A sequence containing a recombinant virus genome is inserted into a vector and transfected with a host cell, so that a recombinant virus capable of efficiently and stably expressing luciferase can be saved, and the luciferase recombinant virus strain can be used for establishing a rapid and high-throughput antiviral drug screening technology or platform by virtue of a mouse bioluminescence in-vivo imaging system. Wide application values are provided for living animal imaging, drug screening, drug action mechanism, vaccine evaluation and the like.

Description

technical field [0001] The invention relates to the technical field of biology, in particular to a lymphatic choroid plexus meningitis virus expressing luciferase gene and its construction method and application. Background technique [0002] Lymphocytic choriomeningitis virus (LCMV) belongs to the genus Mammalian Arenaviridae of the Arenaviridae family and is a negative-sense segmented RNA virus wrapped in an envelope. Armstrong et al discovered and isolated the virus in samples of meningitis outbreak in Louisiana in 1933. The genome consists of a large fragment L (7.2kb) and a small fragment S (3.4kb), in which the L fragment encodes the viral RNA-dependent RNA polymerase L (~200kDa) and the matrix protein Z (~11kDa), and the S fragment encodes the viral Nucleoprotein NP (-63 kDa) and envelope glycoprotein and envelope glycoprotein GPC (-75 kDa). There is a UTR (Untranslated region) sequence at the 5' and 3' ends of each genome, and the UTRs at both ends can be complemen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/65C12N15/64C12N7/01C12Q1/02A61K49/00
CPCC12N15/86C12N15/65C12N15/64C12N7/00C12N9/0069G01N33/5008A61K49/0008A61K49/0045C12N2760/10043C12N2760/10021G01N2500/10
Inventor 张磊砢肖庚富尚卫娟温宇稀徐欢万伟玮
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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