A method of silencing the ifnar2 gene in the df‑1 cell line

A cell line, DF-1 technology, applied in DNA/RNA fragmentation, recombinant DNA technology, genetic engineering and other directions, can solve the problems of insufficient antigen quality and high production cost of vaccines, and solve the problem of insufficient antigen quality and high production cost. , the effect of reducing production costs

Active Publication Date: 2018-04-17
山东英科维芯生物工程有限公司
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Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiencies existing in the production of existing avian influenza vaccines, to provide a method for silencing the IFNAR2 gene in the DF-1 cell line, which can increase the antigen content of the avian influenza virus in the DF-1 cell line to 5- 10 times, not only can solve the problem of insufficient vaccine antigen and high production cost, but also promote the research and development of bird flu bioreactor cell culture technology

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  • A method of silencing the ifnar2 gene in the df‑1 cell line
  • A method of silencing the ifnar2 gene in the df‑1 cell line
  • A method of silencing the ifnar2 gene in the df‑1 cell line

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Embodiment Construction

[0032] 1. Screening of siRNA that interferes with IFNAR2 gene transcription

[0033] 1. Design and synthesis of oligonucleotide sequence of siRNA interfering with IFNAR2 gene

[0034] According to the IFNAR2 gene sequence registered in Genbank and the addgene lentiviral vector construction program, siRNAs with 3 RNA interference target sequences were designed at http: / / jura.wi.mit.edu / bioc / siRNAext / (such as figure 1 shown), and set up the control group Scramble at the same time, the sequence is as follows: sh1: 5′-AATAACCTCTGTAGAGATCAT-3′ (SEQ No.1); sh2: 5′-AA AGACACGGATAGTGAGTTA-3′ (SEQ No.2); sh3: 5′- TACACAAGGCGTGATATCGTA-3'(SEQ No.3); Scramble: CCTAAGGTTAAGTCGCCCTCG(SEQ No.4), according to the characteristics of the pLKO.1-TRC cloning vector, the DNA sequence of the siRNA and its corresponding complementary sequence were connected with a loop sequence to form a sense strand and a reverse The sense strand is introduced into the adapter sequence at both ends, and shRNA is...

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Abstract

The invention discloses a method for silencing IFNAR2 gene in DF-1 cell line. It firstly designs multiple siRNAs with RNA interference target sequences based on the IFNAR2 gene, and further screen out siRNAs that knock down the expression of the IFNAR2 gene at the mRNA level; then construct a lentiviral vector containing the IFNAR2 RNAi gene; through the above lentiviral vector, the IFNAR2RNAi Gene-mediated introduction into DF-1 cell line silences the expression of IFNAR2 gene and increases the proliferation titer of avian influenza virus in DF-1 cell line. This method can increase the antigenic quantity of avian influenza virus in the DF-1 cell line by 5-10 times, greatly reducing the cost of vaccine production. The development process of organ cell culture technology.

Description

technical field [0001] The invention relates to a method for silencing an interferon receptor 2 (Interferon receptor 2, IFNAR2) gene in a DF-1 cell line, which is used for increasing the proliferation titer of avian influenza virus in the cell line, and belongs to the field of biotechnology. Background technique [0002] With the rapid development of intensive breeding industry, important poultry diseases such as avian influenza are becoming the focus of public health security, and controlling diseases from the source is the top priority of the current prevention and control strategy. Practice has proved that vaccine immunization and rapid detection are the most effective measures to prevent and control major animal diseases. Due to the low titer of avian influenza virus cell culture, poultry embryos are still mainly used to produce vaccines in China at present, which has low efficiency, high energy consumption, and the possibility of foreign virus contamination. my country...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/12C12N15/113
Inventor 黄兵刘存霞马秀丽艾武刘爱玲李玉峰刘华雷宋敏训王月明刘洪对
Owner 山东英科维芯生物工程有限公司
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