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Reporter gene plasmid for testing RNA (Ribonucleic Acid) polymerase activity of influenza viruses in canine cells and application thereof

A technology of RNA polymerase and influenza virus, which is applied in the direction of recombinant DNA technology, microbe-based methods, microbiological measurement/inspection, etc., can solve the problems of inconvenient operation and inability to accurately reflect the activity of viral polymerase polymerase, etc., and achieve The detection method is simple and the effect of a wide range of applications

Inactive Publication Date: 2014-01-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional determination of influenza virus polymerase activity uses a dual reporter gene detection system. The reporter genes are renilla luciferase and firefly luciferase respectively. Since renilla luciferase and firefly luciferase cannot be secreted outside the cell, they need to be lysed. Cells are used to measure the expression level of luciferase, which is inconvenient to operate; in addition, most of the reporter gene plasmids used in the past are human-derived promoters, which cannot accurately reflect the polymerase activity of viral polymerases on canine cells

Method used

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  • Reporter gene plasmid for testing RNA (Ribonucleic Acid) polymerase activity of influenza viruses in canine cells and application thereof
  • Reporter gene plasmid for testing RNA (Ribonucleic Acid) polymerase activity of influenza viruses in canine cells and application thereof
  • Reporter gene plasmid for testing RNA (Ribonucleic Acid) polymerase activity of influenza viruses in canine cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Construction of pGLucCa plasmid

[0043] 1. Construction of pFluGLuc plasmid

[0044] (1) Synthesis of gene fragment A

[0045] 1. Refer to the pRSET-TAUT_2P sequence (ID: gb|KC795008.1|) provided by NCBI to determine the T7 promoter sequence as 5’-TAATACGACTCACTATAGG-3’;

[0046] 2. With reference to the 28S ribosome termination region sequence (ID: gb|M12074.1|), determine the reverse complement of the RNA polymerase I terminator sequence as 5’-ccggagtactggtcgacctccgaagttggggggg-3’;

[0047] 3. Refer to the influenza virus database provided by NCBI to determine the 5'UTR conservative sequence of the nonstructural protein NS1 gene as 5'-AGCAAAAGCAGGGTGACAAAAACATA-3'.

[0048] 4. According to the T7 promoter, the reverse complementary sequence of the RNA polymerase Ⅰ terminator sequence and the 5'UTR conservative sequence of the non-structural protein NS1 gene, the gene fragment A is synthesized, and the gene sequence is

[0049] 5’-TTG Gaattc taatacgactcactatagggagaccc...

Embodiment 2

[0064] Example 2. Determination of influenza virus RNA polymerase activity in canine cells

[0065] 1. Extract swine H1N2 influenza virus A / swine / Guangdong / 1222 / 2006 (H1N2), avian H9N2 influenza virus A / chicken / Hebei / LC / 2008 (H9N2) and human H1N1 influenza virus A / Puerto Rico / 8 / 34( H1N1) RNA and reverse transcribed into cDNA to obtain cDNA of swine H1N2 influenza virus, avian H9N2 influenza virus cDNA and human H1N1 influenza virus cDNA.

[0066] 2. Synthesize the primers in Table 1.

[0067] Table 1 PCR primers

[0068]

[0069] Three, plasmid construction

[0070] (1) PCR was performed with the cDNA of the swine H1N2 influenza virus as a template and PB2-1 and PB2-2341R as primers to obtain the PB2 gene of the swine H1N2 influenza virus; EcoRV and XhoI double enzyme digestion of the PB2 gene to obtain a gene fragment; EcoRV and The pcDNA3.1(+) plasmid was digested with XhoI to obtain a large vector fragment; the gene fragment was connected with the large vector fragment to obtain a ...

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Abstract

The invention discloses a reporter gene plasmid for testing RNA (Ribonucleic Acid) polymerase activity of influenza viruses in canine cells and application thereof. According to the plasmid disclosed by the invention, the nucleotide sequence is shown in SEQ ID No. 1. In a dual-reporting genetic testing method, by adopting the reporter gene plasmid pGLucCa constructed by the invention, the RNA polymerase activity of the influenza viruses in the canine cells can be accurately reflected, errors caused by lysed cells can be reduced, cell supernatant can be extracted at different time points so as to carry out RNA polymerase activity assay, and then, the dual-reporting genetic RNA polymerase activity testing method is simpler and more convenient and is wider in range of application.

Description

Technical field [0001] The invention relates to a reporter gene plasmid for detecting influenza virus RNA polymerase activity in canine cells and its application. Background technique [0002] Canine influenza is a highly contagious infection in dogs caused by the canine influenza virus. Suffering dogs often show cold symptoms such as cough and pneumonia. In recent years, reports of canine influenza virus infection have increased rapidly. Dogs are important companion animals for humans, and the health of dogs has attracted more and more attention. In addition, influenza viruses in dogs may increase the chance of infecting people through close human-dog contact. Therefore, canine influenza virus not only affects the health of dogs, but also poses a serious threat to public health safety. [0003] The polymerase gene of influenza virus plays an important role in its pathogenicity, host adaptability and inter-species transmission and other biological characteristics. Studies have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12Q1/68C12Q1/66C12Q1/42C12Q1/48C12R1/93
Inventor 孙怡朋刘金华刘林青
Owner CHINA AGRI UNIV
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