Nanometer antibody for avian influenza virus H7N2, and application of nanometer antibody

A technology of nano-antibody and influenza A virus, which is applied in the field of biopharmaceuticals, can solve the problems of pathogenicity, and there is no report on the epitope nano-antibody of influenza A H7N2 virus, and achieve a good linear relationship

Active Publication Date: 2016-12-07
北京科卫临床诊断试剂有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All H7 lineages with different antigenicities can cause human disease. Human isolates of North American lineage H7N2 and H7N3 and Eurasian lineage H7N2 and H7N3 isolated from the population in recent years can be used as suitable vaccine strains, but for the two lineages of human H7 vaccines are still in development
There is no report of nanobodies against epitopes of influenza A H7N2 virus

Method used

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  • Nanometer antibody for avian influenza virus H7N2, and application of nanometer antibody
  • Nanometer antibody for avian influenza virus H7N2, and application of nanometer antibody
  • Nanometer antibody for avian influenza virus H7N2, and application of nanometer antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of heavy chain antibody VHH library against influenza A virus H7N2

[0028] (1) Bactrian camels were immunized with inactivated influenza A H7N2 virus, each time the inactivated influenza A H7N2 virus was mixed with Freund's adjuvant in a volume of 1:1, once a week, a total of 7 times of immunization, the first Complete Freund's adjuvant was used for the first time, and Freund's incomplete adjuvant was used for the remaining six times to stimulate B cells to express the heavy chain antibody VHH specific to influenza A virus H7N2 during the immunization process. (2) After the 7 times of immunization, 100 ml of camel peripheral blood lymphocytes were extracted and total RNA was extracted. (3) The extracted RNA was reverse-transcribed into cDNA and the VHH chain was amplified by nested PCR. The results were as follows: figure 1 It was shown that the size of the fragment was about 500 bp. (4) Using restriction endonucleases Pst I and not I (p...

Embodiment 2

[0029] Example 2: Screening process for heavy chain antibody VHH against influenza A virus H7N2

[0030] (1) Dissolve in 100 mM pH 8.2 NaHCO 3 Conjugate 200 μg of inactivated influenza A H7N2 virus in NUNC microtiter plates, place overnight at 4°C, and set up a negative control coated with NaHCO 3 . (2) On the second day, add 100 μg of 1% skimmed milk to the two wells respectively, and block for 2 hours at room temperature. (3) After 2 hours, add 100 μl of phage and let it react for 1 hour at room temperature. (4) Wash 5 times with PBST (0.05% Tween 20 in PBS) to wash away phages that cannot bind to antigen. (5) Use 100 mM triethanolamine to elute the phages that specifically bind to influenza A virus H1N1, and infect Escherichia coli TG1 in logarithmic phase growth, produce and purify the phages for the next round of screening, the same The screening process was repeated for 3-4 rounds. In the process of continuous screening, positive clones will be continuously enriche...

Embodiment 3

[0031] Example 3: Using phage enzyme-linked immunosorbent method (ELISA) to screen specific single positive clones:

[0032] (1) From the cell culture dish containing phage after the above 3-4 rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 μg / ml ampicillin (1L TB medium contains 2.3 g diphosphate Potassium hydrogen phosphate, 12.52 g dipotassium hydrogen phosphate, 12 g peptone, 24 g yeast extract, 4 mL glycerol), after growing to the logarithmic phase, add IPTG at a final concentration of 1 mM, and culture overnight at 28°C. (2) Use the osmotic pressure method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour. (3) Wash off unbound antibodies with PBST, add primary mouse anti-HA tag antibody (anti-mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), and place at room temperature for 1 hour. (4) Wash of...

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Abstract

The invention discloses a nanometer antibody in accordance with epitope of avian influenza virus H7N2, and a gene sequence encoding the nanometer antibody, and besides, further discloses a host cell capable of expressing the nanometer antibody for the influenza virus H7N2. The nanometer antibody for the avian influenza virus H7N2 and the encoding sequence of the nanometer antibody are obtained for the first time, the nanometer antibody can be efficiently expressed in escherichia coli, can specifically recognize the avian influenza virus H7N2, is high in detection sensitivity, presents favorable linear relationship when being used for detecting the avian influenza virus H7N2, and has the application value of diagnosing the avian influenza virus H7N2.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a nanobody against avian influenza A H7N2, its coding sequence and its application. Background technique [0002] The genome of influenza A virus (Avian Influenza Virus, AIV) is a negative-strand RNA of eight segments, belonging to the Orthomyxoviridae family, which encodes 12-13 proteins, according to the two surface proteins HA (hemagglutinin) and NA (Neuraminidase) can be divided into different serotypes, and 81 different serotypes of avian influenza viruses have been isolated from poultry, and new HA and NA are constantly being discovered. Sexuality brings great difficulty to the prevention and control of bird flu. According to the degree of pathogenicity, avian influenza viruses can be divided into three types: highly pathogenic (HPAIV), low pathogenic (LPAIV) and non-pathogenic. Non-pathogenic avian influenza will not cause obvious symptoms; low pathogenicity H...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21G01N33/569C12R1/19
Inventor 万亚坤龚雪欧卫军王保君
Owner 北京科卫临床诊断试剂有限公司
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