Novel anti-H5 subtype avian influenza virus nanoantibody and application thereof

An avian influenza virus and nanobody technology, used in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of long development cycle of monoclonal antibodies, difficult to obtain antibodies, and small size, and achieve good stability and biological stability. Chemical activity, improved accuracy and reproducibility, and high protein purity

Active Publication Date: 2021-10-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To quickly and accurately diagnose influenza virus subtypes, a large number of studies have reported various methods for detecting influenza viruses, such as virus isolation, immunofluorescence, polymerase chain reaction (PCR), enzyme staining immunosorbent assay (ELISA) and Serological methods, however, these methods are time-consuming and labor-intensive, requiring high cost in addition to laboratory equipment
For example, virus isolation is considered the gold standard for diagnosis and is essential for routine rapid laboratory confirmation of human influenza, but it often requires 5–7 days of testing with labor-intensive and long procedures; while molecular detection method

Method used

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  • Novel anti-H5 subtype avian influenza virus nanoantibody and application thereof
  • Novel anti-H5 subtype avian influenza virus nanoantibody and application thereof
  • Novel anti-H5 subtype avian influenza virus nanoantibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The recombinant AIV inactivated vaccine (H5N1 subtype, RE-8 strain) avian influenza virus is immunized, once per three weeks, a total of immunization, extracting the peripheral lymphocyte of the immunization, separating the peripheral blood Lymphocyte (PBMC) was extracted for RNA, and the reverse transcription was obtained, and the VHH fragment was obtained by expansion of the second neocra PCR, and the VHH fragment was connected to the PCANTAB-5E phage vector, and the product was converted to XL- BLUE, constructs anti-protocarically targeted nano antibody libraries; assisted phage VCSM13 superfeiteria XL-BLUE bacteria, after overnight culture, collected upper and upper level can be obtained to obtain phage nanocarbon libraries.

Embodiment 2

[0061] The inactivated antigen H5 subtype avian flu (RE-8 strain) package was used on the enzyme label, 100 μL per hole (0.05 mol / l, pH 9.6). 10%, placed in 37 ° C for 2 h; discarded liquid, washed with 100 μl of PBS, add 300 μL of 3% BSA to be placed at room temperature to block 1h; discard the sealing liquid, add 100 μl of phage antibody fluid, incubate at room temperature for 1 h; Liquid and unbonded phage were washed 5 times with 0.05% PBST, 5 min / times, and 100 μl of glycine-hydrochloric acid pH = 2.2 was added, and the eluate was collected, and the amount of 2 mTris base was added, so that the liquid containing a phage In neutrality, the first round of enrichment screening phage; the phage is further amplified, and the next round of screening; the five-wheel "adsorption-eluting-amplification" screening process can eventually achieve strong specificity The phage clone (wherein the first round of screening adopts non-specific conditions, the latter round screening is impro...

Embodiment 3

[0063] Choosing a single colonite for ELISA method screening positive clone: ​​a single colony is closed by a method package by the package to the enzyme label, with a blank control, and then add 100 μL / hole. HRP-M13, 37 ° C was incubated for 1 h, washed 5 times with 0.05% PBST, 5 min / times, add 100 μl / hole TMB substrate color color, and the protected room temperature was placed for 20 min, and the enzyme gauge was 620 nm OD value, with P / N value (i.e., the ratio of the positive hole OD reading / control hole OD reading) is greater than or equal to 3 is positive. The sequencing of positive clones can be obtained to obtain an anti-H5 subtype avian influenza nanobody sequence.

[0064] Its amino acid sequence is shown in SEQ ID NO.1: including the frame area (FR) and an antibody gene complementary determining region (CDR), the framework region is divided into four parts, sequentially defines FR1, FR2, FR3, FR4, sequentially recorded as SEQ ID No. 2, SEQ IDNO.3, SEQ ID No.4, ...

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Abstract

The invention discloses a novel anti-H5 subtype avian influenza virus nanoantibody and application thereof. The amino acid sequence of the nanoantibody is as shown in SEQ ID NO. 1. An antibody gene is connected with a maltose binding protein (MBP) gene through a connecting peptide, an expression vector is constructed, and the expression of a fusion protein is induced to promote the soluble expression of the anti-H5 avian influenza virus nanoantibody fusion protein in an expression system, so the problem that the anti-H5 avian influenza virus nanoantibody protein is insoluble in a prokaryotic system is solved, and accuracy and repeatability are improved. The nanoantibody can be used for detecting, diagnosing, preventing or treating avian influenza viruses, especially H5 subtype avian influenza viruses. The nanoantibody has great significance for monitoring the H5 avian influenza of chicken.

Description

Technical field [0001] The invention belongs to the field of biomedical technology, and in particular, the present invention relates to an anti-H5 subtype avian influenza virus nano-antibody and its application thereof. Background technique [0002] Avian influenza virus (AIV) belongs to the nominal virus-based sputum, is a class of single-stranded negative segment RNA viruses, depending on influenza nucleus (NP) and matrix protein (M) Different are divided into A, B and C type, also known as A, B and Class C between China. According to the antigenic differences of hemagglutinin (HA) and neuraminidase (Na), the antibody virus can be divided into different subtypes. So far, there are 17 antigens that have different ephemeurized hemagglutinoids, which are further classified as the first group or the second group of hemaglasses (first group: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 viruses; the second group: H3, H4, H7, H10, H14, H15 virus). [0003] Hematochemical (HA) is...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K19/00C12N15/13C12N15/62A61K39/42A61P31/16
CPCC07K16/1018A61P31/16A61K2039/505A61K2039/552C07K2317/565C07K2317/567C07K2319/00
Inventor 许斯祺潘志超廖明冯赛祥张奇谢倩梅代绘琳江金飞陈爱华罗开健
Owner SOUTH CHINA AGRI UNIV
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