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

A technology of RNA polymerase and influenza virus, which is applied in the direction of recombinant DNA technology, measurement/inspection of microorganisms, and the introduction of foreign genetic material using vectors, etc. It can solve the problems of inability to accurately reflect the activity of virus polymerase polymerase and inconvenient operation. , to achieve the effect of simple detection method and wide application range

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

Problems solved by technology

[0005] The traditional determination of influenza virus polymerase activity uses a dual reporter gene system. The reporter genes are renilla luciferase and firefly luciferase respectively. Since renilla luciferase and firefly luciferase cannot be secreted outside the cell, the cells need to be lysed It is inconvenient to measure the expression level of luciferase; in addition, most of the reporter gene plasmids used in the past are human promoters, which cannot accurately reflect the polymerase activity of viral polymerase on poultry cells

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the construction of pGLucCk plasmid

[0044] 1. Construction of pFluGLuc plasmid

[0045] (1) Synthesis of gene fragment A

[0046] 1. Referring to the pRSET-TAUT_2P sequence provided by NCBI (ID: gb|KC795008.1|), determine the T7 promoter sequence as 5'-TAATACGACTCACTATAGG-3';

[0047] 2. Referring to the 28S ribosome termination region sequence (ID: gb|M12074.1|), determine the reverse complementary sequence of the RNA polymerase I terminator sequence as 5'-ccggagtactggtcgacctccgaagttggggggg-3';

[0048] 3. Referring to the influenza virus database provided by NCBI, the conserved sequence of the 5'UTR of the nonstructural protein gene NS1 was determined to be 5'-AGCAAAAGCAGGGTGACAAAACATA-3'.

[0049] 4. According to the reverse complementary sequence of T7 promoter, RNA polymerase I terminator and NS1 gene 5'UTR conservative sequence, synthesize gene fragment A, its sequence is as follows:

[0050] 5'-TTG Gaattc taatacgactcactatagggagacccaagctgttaac...

Embodiment 2

[0069] Embodiment 2, the mensuration of influenza virus RNA polymerase activity in poultry cell

[0070] 1. Extraction of porcine 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 porcine H1N2 influenza virus, cDNA of avian H9N2 influenza virus and cDNA of human H1N1 influenza virus.

[0071] 2. Synthesize the primers in Table 1.

[0072] Table 1 PCR primers

[0073]

[0074]

[0075] 3. Plasmid construction

[0076] (1) Using the cDNA of porcine H1N2 influenza virus as a template and using PB2-1 and PB2-2341R as primers to perform PCR, the PB2 gene of porcine H1N2 influenza virus was obtained; EcoRV and XhoI double-digested the PB2 gene to obtain gene fragments; EcoRV and XhoI double-digested the pcDNA3.1(+) plasmid to obtain a large fragment of the vector; the gene fragment was connected to ...

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Abstract

The invention discloses a reporter gene plasmid for testing RNA (Ribonucleic Acid) polymerase activity of influenza viruses in avian 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 pGLucCk constructed by the invention, the RNA polymerase activity of the influenza viruses in the avian 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 the activity of influenza virus RNA polymerase in poultry cells and its application. Background technique [0002] Avian influenza, the full name of avian influenza, is an animal infectious disease caused by a virus, usually only infecting birds, and rarely infecting pigs. Avian influenza viruses are highly species-specific, but in rare cases can cross the species barrier to infect humans. According to the different types of pathogens, avian influenza can be divided into three categories: highly pathogenic, low pathogenic and non-pathogenic avian influenza. Non-pathogenic avian influenza does not cause obvious symptoms, but only produces virus antibodies in infected birds; low-pathogenic avian influenza can cause mild respiratory symptoms, reduced food intake, decreased egg production, and sporadic deaths in birds; Highly pathogenic avian influenza is the most serious, with high morbidity...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12Q1/68C12Q1/66C12Q1/48C12Q1/42
Inventor 孙怡朋刘林青刘金华
Owner CHINA AGRI UNIV
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