Single-chain antibody for resisting influenza viruses, preparation method for single-chain antibody, application of single-chain antibody

An anti-influenza virus and single-chain antibody technology, applied in the field of bioengineering, can solve the problems of unreported patents, unreported influenza virus single-chain antibody ScFv, etc.

Inactive Publication Date: 2012-07-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, there is no domestic report on the research on the single-chain antibody ScFv o

Method used

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  • Single-chain antibody for resisting influenza viruses, preparation method for single-chain antibody, application of single-chain antibody
  • Single-chain antibody for resisting influenza viruses, preparation method for single-chain antibody, application of single-chain antibody
  • Single-chain antibody for resisting influenza viruses, preparation method for single-chain antibody, application of single-chain antibody

Examples

Experimental program
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preparation example Construction

[0077] Preparation method of single chain antibody

[0078] The method for preparing the anti-influenza virus single-chain antibody of the present invention can be artificial synthesis, which can be referred to various documents, such as liquid phase synthesis or solid phase synthesis of polypeptides. It can also be prepared by using bioengineering methods to express the single-chain antibody of the present invention, and produced from the expressor. It includes transforming, transducing or transfecting host cells with the expression vector, culturing the host cells, isolating proteins from the culture, and obtaining anti-influenza virus single-chain antibodies. Wherein, the recombinant expression transformant is the same as the above introduction, and the present invention can be obtained by transforming the recombinant expressor of the present invention into a host microorganism. Wherein, the medium used in the culture of the recombinant expression transformant can be any m...

Embodiment 1

[0084] Example 1 Extraction of Total RNA of Splenocytes of Immunized Mice and Reverse Transcription to Synthesize Complementary DNA

[0085] 1.1 Extraction of total RNA from splenocytes of mice infected with A / PR / 8 / H1N1 influenza virus

[0086] Get the antigenic influenza virus (influenza virus PR8 (A / Puerto-ico / 8 / 34 (H1N1), which is a virus commonly used in the laboratory, and its biological characteristics can represent the characteristics of the H1N1 influenza virus. Influence of Influenza Virus A / PR / 8 / H1N1 Model Mouse Macrophage Phagocytosis, Journal of Shandong University of Traditional Chinese Medicine, Volume 34, Issue 5, September 2010, Pages 457-458.) Culture supernatant, the The virus liquid was inoculated on MDCK cells (purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and the cell supernatant was drawn after 48 hours, which was the cell culture supernatant containing the virus. ...

Embodiment 2

[0090] Example 2 Synthesis of single-chain antibody gene fragments by overlap extension method

[0091] 2.1 Amplification of the full set of antibody variable region genes

[0092] The cDNA was used as a template to carry out PCR reaction to amplify the heavy chain and light chain variable regions of the antibody gene respectively. The specific steps are as follows: add the following reagents to a 0.2ml PCR tube to amplify the variable region of the heavy chain: 4 μL of immune spleen cell cDNA; 5 μL of 10×PCR buffer; 5 μL of dNTP (10 mM); 1 μL of upstream and downstream primers; Taq (5U / μL ) 0.5 μL, add 50 μL of sterilized ultrapure water. In another PCR reaction tube, add the following reagents to amplify the light chain variable region: 4 μL of immune splenocyte cDNA; 5 μL of 10×PCR buffer; 5 μL of dNTP (10 mM); 1 μL of upstream and downstream primers; 0.5 μL of Taq (5 μ / μL), Add 50 μL of sterilized ultrapure water. The liquid was mixed slowly with a micropipette and cent...

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Abstract

The invention discloses a single-chain antibody ScFv for resisting influenza viruses, a preparation method for the single-chain antibody ScFv, application of the single-chain antibody ScFv, a gene for encoding the single-chain antibody ScFv, a carrier containing the gene, a host cell and the like. The single-chain antibody ScFv for resisting the influenza viruses is one of the following proteins: 1) a single-chain antibody formed by connecting a heavy chain variable region and a light chain variable region of the antibody through a linker peptide, wherein an amino acid sequence of the light chain variable region and an amino acid sequence of the heavy chain variable region are shown as SEQ ID NO.1 and SEQ ID NO.2 in a sequence table respectively; and 2) a derived antibody obtained by improving the single-chain antibody in step 1), wherein the improvement comprises the deletion, substitution or insertion of amino acid, and the derived antibody has the antibody activity of resisting H1N1 influenza viruses. The molecular weight of the single-chain antibody is about 27kD; and the single-chain antibody can specifically identify the H1N1 influenza viruses and block the combination of the viruses and natural serum. The single-chain antibody can be used for diagnosing, preventing, controlling and treating the infection of the H1N1 influenza viruses, or is used for antiviral breeding of transgenic animals.

Description

technical field [0001] The invention belongs to the field of biological engineering. The invention relates to an anti-H1N1 influenza virus single-chain antibody ScFv and its preparation method and application, as well as a gene encoding the single-chain antibody, a vector containing the gene, a host cell and the like. Background technique [0002] Type A influenza virus can infect various poultry and mammals other than humans, such as pigs, horses and marine mammals, and cause acute respiratory infectious diseases. Its epidemic will greatly endanger human health and cause huge losses to the world economy. One of the group diseases that are difficult to cure. According to the difference in antigenicity of hemagglutinin protein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) protein, 16 subtypes and 9 subtypes have been identified so far. At present, at least dozens of influenza viruses with different serological subtypes, such as H1N1, H1N2, H1N7, H2N3, H3N2, H4N6...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/63C12N1/21A61K39/42A61P31/16C12R1/19
Inventor 蒋蔚王权陈永军刘迎春史子学马志永顾惠明杨康宋宁宁石金磊李欣彤
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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