CN112079917A - H7N9 virus specific recognition antibody P51H08 and detection kit - Google Patents

H7N9 virus specific recognition antibody P51H08 and detection kit Download PDF

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CN112079917A
CN112079917A CN202010903175.9A CN202010903175A CN112079917A CN 112079917 A CN112079917 A CN 112079917A CN 202010903175 A CN202010903175 A CN 202010903175A CN 112079917 A CN112079917 A CN 112079917A
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袁冰
徐国东
幸晓莹
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Canvest Wuhan Biotechnology Co ltd
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
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    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
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Abstract

The invention discloses an H7N9 virus specific recognition antibody P51H08 and a detection kit, wherein in the amino acid sequence of the antibody, a heavy chain CDR1: GFTFSSYA, a CDR2: ISGSGGST, a CDR3: AKDLNWWPDY, a light chain CDR1: NIGSKR, a CDR2: NDN and a CDR3: LVWDSSSLHRV are included. The antibody can specifically recognize the H7N9 influenza virus antigen, has high affinity and high specificity, can effectively solve the common false positive condition in the current clinical diagnosis, and provides an accurate detection result for the prevention and control of the influenza virus.

Description

H7N9 virus specific recognition antibody P51H08 and detection kit
Technical Field
The invention relates to the technical field of virus diagnosis, in particular to a specific recognition antibody P51H08 of H7N9 avian influenza virus and a detection kit of the virus.
Background
Acute respiratory infections are the most common infectious diseases in humans. Most respiratory infections are caused by viruses, and clinically common pathogens are adenovirus, influenza virus, parainfluenza virus, respiratory syncytial virus and rhinovirus, while mycoplasma and chlamydia are respiratory pathogenic bacteria. Because of the different treatment regimens taken for different bacterial and viral infections, it is desirable to make a diagnosis as soon as possible after infection. Meanwhile, due to the strong transmission and high lethality of highly pathogenic influenza viruses, strict isolation and protection are required. Since the general clinical diagnosis method cannot identify the difference of pathogens, an accurate and reliable laboratory method is required for the differential diagnosis of pathogens.
To achieve detection of influenza virus antigens, high affinity antibodies against influenza virus are required, and many of the detection antibodies currently on the market are of animal origin, for example: the hybridoma technology develops murine antibodies and rabbit antibodies. However, due to the complexity of human serum components, false positive detection is easily caused, namely, antibodies against murine or rabbit antibodies may still exist in serum without virus infection, and the result of serological diagnosis is false positive. This will cause great difficulty in diagnosing suspected cases that are not clinically developed. In order to solve the problem, the prior scheme is to perform species change on the obtained murine antibody or rabbit antibody, namely to perform humanization on the antibody, but in the process, the affinity and the specificity of the antibody are seriously lost with a high probability, and the finally obtained humanized antibody also loses the application value. Therefore, we used phage display technology to screen human monoclonal antibodies with high affinity and high specificity against influenza virus antigens for clinical diagnosis.
Disclosure of Invention
The research screens the human monoclonal antibody with high affinity and high specificity aiming at the influenza virus H7N9 antigen by constructing a phage display library of the human antibody, is used for clinical diagnosis, solves the common false positive condition in the current clinical diagnosis and provides an accurate detection result for the prevention and control of the influenza virus.
The technical scheme of the invention is detailed as follows:
in a first aspect, the present invention provides an H7N9 virus-specific recognition antibody P51H08, comprising the following amino acid sequences of complementarity-determining regions (CDRs) of the heavy and light chains:
Figure BDA0002660474490000021
the complementarity determining regions are hypervariable regions which are the binding sites of antibodies to antigens, and CDR3 has a higher degree of hypervariability, and the heavy chain plays an important role in antigen binding.
Preferably, in the antibody P51H08, the heavy chain amino acid sequence is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2.
In a second aspect, the present invention provides a gene encoding the above-described antibody P51H 08.
Preferably, in the above coding gene, the heavy chain nucleotide sequence is shown as SEQ ID NO.3, and the light chain nucleotide sequence is shown as SEQ ID NO. 4.
In a third aspect, the present invention provides a vector comprising the above-described encoding gene.
In a fourth aspect, the present invention provides a host cell comprising the above-described encoding gene.
In a fifth aspect, the invention provides an application of the antibody P51H08 in preparing a diagnostic reagent of H7N9 virus.
In a sixth aspect, the invention provides a kit for detecting the specificity of the H7N9 virus, which comprises the antibody P51H 08.
Preferably, the detection kit further comprises a microplate coated with the antibody P51H08, and the modified antibody P51H08 as a secondary antibody, a buffer solution, a sample diluent, a developing solution, a negative control serum and a positive control solution. The buffer solution, the sample diluent, the developing solution, the positive control solution and the like are all conventional reagents in the field, and no special component is required. For example, PBS may be used as the buffer solution and the sample diluent, and HRP-labeled secondary antibody may be used, and TMB color developing solution or ABTS color developing solution may be used. The positive control solution contained H7N9 inactivated antigen and PBS.
Compared with the prior art, the invention has the following beneficial effects:
the antibody P51H08 is a human monoclonal antibody, can specifically recognize H7N9 influenza virus antigen, has high affinity and high specificity, and is combined with EC50The value is less than 1nM, far higher than that of most of the antibodies (EC) at present50The value is generally fifty to hundreds of nanomoles), the affinity is very high, compared with the existing RT-PCR detection method, the direct immunoassay using the antibody is simpler, quicker and lower in cost, the false positive condition commonly existing in the current clinical diagnosis can be effectively solved, and the accurate detection result is provided for the prevention and control of the influenza virus.
In addition, the antibody of the invention is used for immunodetection, and is simpler and quicker than the prior RT-PCR method for detection.
Drawings
FIG. 1 is the electrophoresis picture of the expression product of P51H08 protein.
FIG. 2 shows the results of affinity assay of P51H08 IgG form antibody by ELISA.
FIG. 3 shows the results of experiments in which serum of patients in convalescent period was combined with SH02 HA strain (A/Shanghai/2/2013H 7N9), 17SF HA strain (A/Guangdong/17SF003/2016), and human H5 rHA monomer protein (SH02 MONO,17SF MONO, and human H5 MONO), and BSA was negative control.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
The techniques used in the following examples are, unless otherwise specified, conventional techniques known to those skilled in the art; the instruments, reagents, etc. used, unless otherwise specified in this specification, are publicly available to those of skill and research in the art.
Example 1 screening of H7N9 Virus-specific recognition antibodies
(1) Construction of phage display library of humanized Fab antibody
The results of the binding experiments of the serum of patients in the convalescent period infected with H7N9 virus and SH02 HA, 17SF HA and human H5 rHA monomer proteins (SH02 MONO,17SF MONO and human H5 MONO) are shown in FIG. 3, which indicates that the serum of patients in the convalescent period contains antibodies capable of binding H7 or H5 antigens, and BSA is used as a negative control and HAs no non-specific binding. The blood of a patient with specific binding is selected as a sample for establishing a library.
Taking patient blood capable of specifically combining with the virus protein as a sample, separating lymphocytes, extracting total RNA, synthesizing cDNA through reverse transcription, taking the cDNA as a template, and amplifying Fab fragments of the antibody by using specific primers. The resulting fragments were ligated to phagemid vectors, electroporated into commercial TG1 competence, and helper phage were added to generate human Fab antibody phage display libraries.
(2) Preliminary screening against the HA antigen of H7N9
HA genes of influenza viruses are constructed on baculovirus expression vectors, envelope protein HA of H7N9 is obtained by SF9 cell expression, and the envelope protein HA is used as a target (target protein) to carry out screening to obtain a plurality of candidate antibodies. Affinity determination (ELISA, biosensor) and epitope identification are carried out on the candidate antibodies, and a plurality of candidate clones are determined for further optimization after comprehensive consideration of a plurality of factors.
(3) Affinity maturation of candidate clones
And (3) randomly mutating by taking the screened candidate antibody as a template to construct a phage display library with mature affinity. And continuously utilizing the target protein for screening, and obtaining the clone with higher HA affinity through several rounds of screening-mutation.
(4) Human antibody affinity and specificity assays
Several candidate clones were picked, ligated into eukaryotic expression vectors, expressed in 293F cells and purified in IgG format. These clones were subjected to affinity assays (ELISA, biosensor), specificity assays (double antibody sandwich to verify different HA antigens and other unrelated antigens).
Finally, an antibody fragment with high specificity and sensitivity is obtained by screening and is named as P51H08, the nucleotide sequences of the heavy chain and the light chain are shown as SEQ ID NO.3-4, and the amino acid sequences of the heavy chain and the light chain are shown as SEQ ID NO. 1-2. The result of the IgG protein expression electrophoresis is shown in FIG. 1, and the P51H08 protein is about 55 KD.
Example 2 detection of the Properties of the antibody P51H08
See FIG. 2 for the results of the affinity assay for the P51H08 IgG antibody by ELISA, where · denotes the 17SF HA strain (A/Guingdong/17 SF003/2016), ■ denotes the SH02 HA strain (A/Shanghai/2/2013H 7N9), and a-tangle denotes the BSA negative control.
The results show that the EC of P51H08 on SH02 HA of two strains of H7N9 and the 17SF HA protein50The values are respectively 1.65nM and 3.56nM, the affinity is higher, and the cross activity to HA protein of H7 subtype is certain; at the same time, the ELISA results showed that the antibody was not bound to control BSA and was not non-specific. The antibody has no neutralizing and inhibiting activity.
EC50(nM) P51H08
SH02 HA strain 1.65
17SF HA strain 3.56
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> Wuhan Jia Chuang Biotechnology Limited
<120> H7N9 virus specific recognition antibody P51H08 and detection kit
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Claims (9)

1. An H7N9 virus specific recognition antibody P51H08, characterized in that the amino acid sequences of the complementarity determining regions of the heavy chain and the light chain are as follows:
Figure FDA0002660474480000011
2. the specific recognition antibody P51H08, according to claim 1, wherein the heavy chain amino acid sequence is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.
3. The gene encoding the antibody P51H08 according to claim 2.
4. The encoding gene of claim 2, wherein the heavy chain nucleotide sequence is represented by SEQ ID No.3 and the light chain nucleotide sequence is represented by SEQ ID No. 4.
5. A vector comprising the gene encoding the gene of claim 3 or 4.
6. A host cell comprising the gene encoding the gene of claim 3 or 4.
7. Use of the antibody P51H08 of claim 1 or 2 in the preparation of a diagnostic reagent for H7N9 virus.
8. A kit for detecting H7N9 virus specificity, comprising the antibody P51H08 of claim 1 or 2.
9. The detection kit of claim 7, further comprising a microplate coated with the antibody P51H08, the modified antibody P51H08 serving as a secondary antibody, a buffer solution, a sample diluent, a developing solution, a negative control serum and a positive control solution.
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US10280212B2 (en) * 2016-06-01 2019-05-07 Panasonic Intellectual Property Management Co., Ltd. Composite comprising antibody capable of binding to intranuclear protein of influenza virus
CN109810189A (en) * 2017-11-21 2019-05-28 中国科学院深圳先进技术研究院 The full human monoclonal antibody 4L3 of anti-H7N9 and its preparation method and application
WO2019147891A1 (en) * 2018-01-26 2019-08-01 Children's Hospital Medical Center Compositions and methods for determining avian influenza virus susceptabilty
WO2020119663A1 (en) * 2018-12-13 2020-06-18 中国科学院深圳先进技术研究院 Anti-h7n9 fully human monoclonal antibody 4e18, preparation method therefor, and application thereof

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US10280212B2 (en) * 2016-06-01 2019-05-07 Panasonic Intellectual Property Management Co., Ltd. Composite comprising antibody capable of binding to intranuclear protein of influenza virus
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