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Anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody as well as preparation method and application thereof

An avian influenza virus and heavy chain antibody technology, applied in the biological field, can solve the problems of low detection sensitivity of antigen components, missed diagnosis, and no very mature products, etc., and achieve the effect of improving early diagnosis and clinical treatment level and high binding ability

Active Publication Date: 2011-10-19
CHINESE NAT HUMAN GENOME CENT AT SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous laboratory-level studies have also shown that the detection sensitivity of antigen components is low, which will lead to serious missed diagnoses, and there are no very mature products available at present.

Method used

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  • Anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody as well as preparation method and application thereof
  • Anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody as well as preparation method and application thereof
  • Anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Vicuna immunity and detection of hemagglutination inhibition titer

[0039] 1. Immunization method: Subcutaneous and intramuscular multi-point injection of vaccine (recombinant avian influenza virus inactivated vaccine: H5N1 subtype, Re-1 strain; Harbin Binveken Biotechnology Development Company) into multiple lumps on the back of the neck, follow up and observe the subcutaneous Inject the absorption of the mass to confirm that the immunization is correct.

[0040] 2. Immunization cycle:

[0041] (1) First immunization: the vaccine injection volume is between 40-120mL;

[0042] (2) Second immunization: one month later, collect antiserum samples and determine the titer; carry out the second immunization, the dose is half of the first;

[0043] (3) The third immunization: collect antiserum samples four weeks later, and measure the titer; and carry out the third immunization, the dose is 1 / 4 of the first time; collect antiserum samples four weeks later, and me...

Embodiment 2

[0055] Example 2: Lymphocyte Separation Process

[0056] 1. Anesthetized animals: before anesthesia, inject 1.0 mL of atropine hydrochloride (Tianjin Pharmaceutical Group Xinzheng Co., Ltd.) into the femoral muscle; inject 1.0 mL of Sumianxin injection (Changchun Military University Veterinary Research Institute) into the femoral muscle, and there is a response within 5 minutes. The effective time of anesthesia is 30 minutes; if it cannot wake up naturally, inject 1 mL of Lu Xingning (Changchun Military University Veterinary Research Institute) to wake up.

[0057] 2. Prepare for blood collection: fast for 12 hours (to prevent vomiting after anesthesia, this step can be saved); take 10-20 mL of blood from the jugular vein, store it at room temperature and away from light.

[0058] 3. Blood sample collection

[0059] A. Non-anticoagulant blood: 5mL blood sample, divided into 5 EP tubes; place it in the dark at room temperature for 2 hours, then place it in a refrigerator at 4°...

Embodiment 3

[0068] Example 3: Extraction of total cellular RNA

[0069] 1. Every 2×10 7 Add 1-2mL Trizol (Invitrogen Company, a new type of total RNA extraction reagent to the cells, which can directly extract total RNA from cells or tissues. It contains phenol, guanidine isothiocyanate and other substances, which can quickly break cells and inhibit the release of cells. nuclease), pipette evenly and mix on a vortex shaker for 30 seconds; let stand at room temperature for 10 minutes (for tissue samples, 1 mL of Trizol can be added per 100 mg of tissue, liquid nitrogen grinding or electric homogenizer fully break up the tissue pieces).

[0070] 2. Add about 1 / 5-2 / 5 volume of chloroform (Sigma company), manually shake up and down vigorously for about 1 minute, and let it stand at room temperature for 5 minutes.

[0071] After centrifuging at 12000rpm for 15min at 3.4°C, carefully remove the supernatant (approximately no more than 3 / 5 of the volume of Trizol), transfer the supernatant to a...

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Abstract

The invention discloses an anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody, which is selected from nucleotide sequences shown by SEQ ID NO:1- SEQ ID NO:10, and nucleotide sequences shown by SEQ ID NO:1- SEQ ID NO:10 are respectively coded and have proteins of nucleotide sequences shown by SEQ ID NO:11- SEQ ID NO:20. In addition, the invention further discloses a preparation method for the anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody, and the method comprises the steps of constructing an anti-H5N1 type bird flue virus vicuna VHH heavy chain antibody library through primer design, PCR (polymerase Chain reaction) conditions, connection and transformation to ensure the capacity and diversity of an antibody library; and then carrying out bacteriophage display from the antibody library through solid phase screening of the optimized antibody library, and screening out 10 phage display antibodies (having nucleotide sequences shown by SEQ ID NO:1- SEQ ID NO:10). In addition, the invention further discloses application of the vicuna VHH heavy chain antibody in H5N1 type bird flu virus lectin haemagglutination inhibition.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a vicuna VHH heavy chain antibody against H5N1 avian influenza virus. In addition, the present invention also relates to the preparation method and use of the antibody. Background technique [0002] Avian influenza is the abbreviation of avian influenza. It is an infectious disease syndrome caused by a subtype of influenza A virus. According to the type of pathogen, avian influenza can be divided into three categories: highly pathogenic, low pathogenic and non-pathogenic. Avian influenza viruses are divided into 15 subtypes such as H1 to H15 according to their surface proteins. Avian influenza around the world is mainly caused by two highly pathogenic subtypes, H5 and H7, while humans are sensitive to H1 and H3. subtype susceptible. According to the statistics of the World Health Organization (WHO), from 2003 to June 20, 2006, a total of 228 people were infected with the H5N1 subt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/12C40B50/06C40B30/04A61K39/42A61P7/02
Inventor 赵国屏王颖熊慧严安包文静金维荣陈志南孙志伟车小燕
Owner CHINESE NAT HUMAN GENOME CENT AT SHANGHAI
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