DNA fragment and application thereof in preparation of H5N1-subtype flu Guassia luciferase reporter virus

An influenza virus and fragment technology, applied in DNA / RNA fragment, recombinant DNA technology, virus / phage and other directions, can solve problems such as application research of unexpressed virus, achieve good objectivity and repeatability, and simplify the process.

Inactive Publication Date: 2015-04-15
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

[0004] At present, there is no recombinant H5N1 subtype influenza reporter virus expressing Gaussia luciferase and its related application research

Method used

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  • DNA fragment and application thereof in preparation of H5N1-subtype flu Guassia luciferase reporter virus
  • DNA fragment and application thereof in preparation of H5N1-subtype flu Guassia luciferase reporter virus
  • DNA fragment and application thereof in preparation of H5N1-subtype flu Guassia luciferase reporter virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, construction of recombinant vector pHW2000-VN-NA-GLUC

[0034] 1. PCR amplification of NA gene coding region (including 3' non-coding region)

[0035] Using the bidirectional expression plasmid pHW2000-VN-NA of the NA segment of the VN1194 virus as a template, use primer pair A (composed of primers A-F and primers A-R, the sequence of primers A-F is shown in Sequence Listing Sequence 5, and the sequence of primers A-R is shown in Sequence Listing Sequence 6) for PCR amplification, and the amplified product was subjected to 1% agarose gel electrophoresis ( figure 1 Swimming lane 1 in ), recovered a 1.4kb DNA fragment (named fragment a) for sequencing, and the results showed that the sequence of fragment a was as shown in sequence 1 in the sequence table, wherein the 1-14th position was the restriction endonuclease Bsa The protected base of I and its recognition site; the 15th-34th position is the 3' non-coding region sequence of the NA segment of the H5N1 sub...

Embodiment 2

[0045] Example 2, Rescue of H5N1 subtype influenza Guassia luciferase reporter virus and identification of luciferase activity

[0046] 1. Virus rescue

[0047] Human renal epithelial cell line HEK293T cells (purchased from ATCC, USA, catalog number CRL-11268) were inoculated in 6-well plates and cultured in high-glucose DMEM containing 10% fetal bovine serum (purchased from Invitrogen, USA, catalog number 1122050) (Invitrogen was purchased from Invitrogen, USA, catalog number 12100-046) for culture. The next day, take the pHW2000-VN-NA-GLUC plasmid and the bidirectional expression plasmids pHW2000-VN-PB2, pHW2000-VN-PB2, pHW2000-VN-PB1, pHW2000-VN-PA, pHW2000- VN-HA, pHW2000-VN-NP, pHW2000-VN-M, pHW2000-VN-NS each 1μg, mix well and add to 500μL opti-MEM culture medium, then add 10μL Lipofectamine LTX (Invitrogen), mix gently , placed at room temperature for 30 minutes to obtain a mixture of plasmids and liposomes. Before transfection, wash the 293T cells once with opti-MEM...

Embodiment 3

[0057] Embodiment 3, growth curve determination

[0058] MDCK cells were seeded in 12-well plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum. The next day, the culture medium was discarded, and the H5N1 subtype influenza Guassia luciferase reporter virus solution and the wild VN1194 virus obtained in step 1 were diluted to a certain concentration with DMEM medium containing 2% fetal bovine serum, and then inoculated at MOI=1 to In cell wells, 200 μl per well, adsorb for 1 hour at 37°C. After that, 800 μl of DMEM medium containing 2% fetal bovine serum was added to each well, and placed at 37°C, 5% CO 2 After culture, the supernatant was collected at 3, 6, 12, 24, and 48 hours after infection, and the virus titer was measured by plaque method, and the correlation between virus titer and Gaussia luciferase activity was calculated by regression analysis.

[0059] The result is as Image 6 As shown, the proliferation ability of the reporter virus...

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Abstract

The invention discloses a DNA fragment and an application thereof in preparation of an H5N1-subtype flu Guassia luciferase reporter virus. The nucleotide sequence of the DNA fragment is composed of following sequences in a successive series manner: an H5N1-subtype flu virus NA segment 3'-terminal noncoded region coding gene sequence, an H5N1-subtype flu virus NA gene encoded region coding sequence, a PTV-1 virus 2A peptide encoded gene sequence, a Gaussia luciferase encoded gene sequence, a H5N1-subtype flu virus NA segment 5'-terminal packaged signal encoded gene sequence, and a H5N1-subtype flu virus NA segment 5'-terminal noncoded region coding sequence. By means of the recombined H5N1-subtype flu reporter virus prepared with the DNA fragments, quantitative detection on viruses in a sample can be objectively carried out quickly and accurately through activity of luciferase in cellular supernatant or animal serum. The H5N1-subtype flu Guassia luciferase reporter virus can not only save time but also improve accuracy and reliability of a detection result, thereby providing a new convenient and effective tool for drug screening and vaccine assessment of anti-H5N1-subtype flu virus drugs.

Description

technical field [0001] The invention relates to a DNA fragment and its application in preparing H5N1 subtype influenza Guassia luciferase reporter virus. Background technique [0002] The H5N1 subtype influenza virus is a type of influenza A virus that is mainly transmitted in birds. In recent years, reports of H5N1 subtype influenza virus infecting humans have increased year by year. As of August 2012, 608 people have been reported infected in 15 countries and regions around the world, of which 359 people died, with a mortality rate as high as 59%. Infection titer is the basic data required for virus-related basic research and drug and vaccine development. At present, the infectious titer of H5N1 subtype influenza virus is mainly determined by measuring half the tissue culture infectious dose (TCID50) or plaque forming unit (PFU) of the virus, the operation is complex and the accuracy is easily affected by subjective experience. [0003] Gaussia luciferase is a newly disc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N7/01
Inventor 户义祝庆余李靖吴晓燕孙伟韩鹏飞杨银辉康晓平李裕昌张雨
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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