CN112876568A - Assembled protein element and application thereof - Google Patents

Assembled protein element and application thereof Download PDF

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Publication number
CN112876568A
CN112876568A CN202110032085.1A CN202110032085A CN112876568A CN 112876568 A CN112876568 A CN 112876568A CN 202110032085 A CN202110032085 A CN 202110032085A CN 112876568 A CN112876568 A CN 112876568A
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protein
complementary
test object
single domain
sample
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耿勇
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Shanghai Yongjian Biotechnology Co ltd
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Shanghai Yongjian Biotechnology Co ltd
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Priority to CN202110032085.1A priority Critical patent/CN112876568A/en
Publication of CN112876568A publication Critical patent/CN112876568A/en
Priority to PCT/CN2022/075886 priority patent/WO2022148491A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis 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
    • G01N2469/10—Detection of antigens from microorganism in sample from host

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a single domain antibody or scFV or Fab fragment of the antibody and complementary fragment of enzyme, which are connected to detect protein, peptide, virus, microbe and other biological substances or biological small molecular substances. Specifically, the invention discloses a detection system which is formed by separating a molecule of biological protease into two or more different fragments, and connecting the fragments with complementary properties with different single domain antibodies or scFV or Fab fragments of the antibodies. In the system, the antibody of the single domain acts on a detected target to assemble enzyme with complementary activity, and the content of the detected biological substance is analyzed by catalyzing a substrate through the activity of the enzyme to form a product, so that the aim of detecting the target is fulfilled.

Description

Assembled protein element and application thereof
Technical Field
The invention relates to detection of various biomolecules in the fields of biomedicine, agriculture, animal husbandry and fishery breeding. In particular to an assembled protein element and application thereof.
Background
Complementary fragments of biological enzymes were ligated to interacting proteins to observe the dynamic properties of the interacting proteins.
Disclosure of Invention
The object of the present invention is a method for the detection and analysis of the content of a biological substance by linking different single domain antibodies or scFV or Fab fragments of antibodies to the complementary fragments of biological enzymes of the biological substance to be detected.
In a first aspect of the invention there is provided an inter-assemblable protein module comprising: n protein elements, wherein n is 2, 3 or 4, each protein element having the structure of formula I,
Pi－(Li－NBi)m(I)
in the formula (I), the compound is shown in the specification,
i is a positive integer from 1 to n;
pi is the i' th complement of an active protein;
li is nothing, a bond or a linker;
NBi is the ith anchor element connected to Pi through Li;
m is 1 to 3;
wherein each Pi is a complementary fragment from the same active protein and each Nbi is independently targeted to and binds to a predetermined assay target;
wherein, when the test object is present, each protein element binds to the test object via its respective anchor element such that all Pi together form a reconstituted complementary fragment complex (i.e., a reconstituted active protein) that has or retains the activity of the active protein;
when the test object is not present, and the protein elements are not bound to the test object, all Pi do not together form the reconstructed complementary fragment complex (i.e., a complex structure with a function cannot be formed).
In another preferred embodiment, NBi (i.e. NB1, NB2, …, NBn) are each independently selected from the group consisting of: single domain antibodies (including, without limitation, nanobodies and artificially synthesized single domain antibodies), scFV, Fab fragments, or combinations thereof.
In another preferred embodiment, NBi is a single domain antibody (or nanobody).
In another preferred embodiment, NBi comprises: (a) a single domain antibody, scFV, Fab fragment, or (b)2 or more (e.g. 2, 3 or 4) single domain antibodies, scFV, Fab fragments in tandem.
In another preferred embodiment, each Li (i.e., L1, L2, …, Ln) is independently absent, a bond, or a linker.
In another preferred embodiment, the joint comprises a flexible structure.
In another preferred embodiment, the linker is a peptide linker.
In another preferred embodiment, the peptide linker comprises 1-50 amino acids, preferably 3-40 amino acids, more preferably 5-30 amino acids.
In another preferred embodiment, when n is 2, the protein module comprises: 2 protein elements, wherein the first protein element has a structure according to formula Ia and the second protein element has a structure according to formula Ib,
P1－(L1－NB1)m(Ia)
P2－(L2－NB2)m(Ib)
in the formula (I), the compound is shown in the specification,
p1 is the 1 st complementary fragment of an active protein;
l1 is nothing, a bond or a linker;
NB1 is anchor element No. 1 connected to P1 through L1;
p2 is the 2 nd complementary fragment of an active protein;
l2 is nothing, a bond or a linker;
NB2 is the 2 nd anchor element connected to P2 through L2;
each m is independently 1 to 3.
In another preferred embodiment, when n is 3, the protein module comprises: 3 protein elements, wherein the first protein element has a structure of formula Ia, the second protein element has a structure of formula Ib, and the third protein element has a structure of formula Ic,
P1－(L1－NB1)m(Ia)
P2－(L2－NB2)m(Ib)
P3－(L3－NB3)m(Ic)
in the formula (I), the compound is shown in the specification,
p1 is the 1 st complementary fragment of an active protein;
l1 is nothing, a bond or a linker;
NB1 is anchor element No. 1 connected to P1 through L1;
p2 is the 2 nd complementary fragment of an active protein;
l2 is nothing, a bond or a linker;
NB2 is the 2 nd anchor element connected to P2 through L2;
p3 is the 3 rd complement of an active protein;
l3 is nothing, a bond or a linker;
NB3 is the 3 rd anchor element connected to P3 through L3;
each m is independently 1 to 3.
In another preferred embodiment, the active protein is selected from the group consisting of: enzymes, interacting protein complexes, small molecule modified interacting proteins capable of generating a signal.
In another preferred embodiment, the active protein is selected from the group consisting of: luciferase, nuclease, endonuclease, protease, and fluorescent protein.
In another preferred embodiment, the complementary fragment complex is capable of catalyzing a reaction on a substrate to produce a detectable signal.
In another preferred embodiment, the detectable signal is selected from the group consisting of: a fluorescent signal, an electrical or chemical signal, or a combination thereof.
In another preferred embodiment, the substrate is selected from the group consisting of: fluorescein, a modified DNA fragment, a modified polypeptide fragment, or a combination thereof.
In another preferred embodiment, the NBi is each independently connected to the N-terminal, C-terminal, middle, or a combination thereof of Pi through Li.
In another preferred embodiment, the predetermined detection object is selected from the group consisting of: proteins, nucleic acids, pathogenic microorganisms (pathogens).
In another preferred embodiment, the pathogen is selected from the group consisting of: viruses, bacteria, chlamydia, fungi, protists.
In a second aspect of the invention there is provided a nucleotide molecule encoding an inter-assemblable protein component according to the first aspect.
In another preferred embodiment, the coding sequence encoding each protein element is located on one or more nucleotide sequences.
In a third aspect of the invention, there is provided an expression vector comprising the nucleotide molecule of the second aspect.
In another preferred embodiment, the coding sequences encoding the respective protein elements are located on one or more expression vectors.
In another preferred embodiment, the expression vector comprises a plasmid or a viral vector.
In a fourth aspect of the invention, there is provided a host cell comprising the expression vector of the third aspect; or the host cell has integrated into its genome the nucleotide molecule of the second aspect.
In a fifth aspect of the invention there is provided the use of the mutually assemblable protein components of the first aspect in the preparation of a protein, peptide, nucleic acid, virus, bacterium, fungus, chlamydia, protist detection reagent or kit.
In a sixth aspect of the present invention, there is provided a detection method comprising:
(a) providing a sample to be tested, wherein the sample may contain a predetermined test object;
(b) mixing said sample with said mutually assemblable protein modules of the first aspect to form a detection system;
(c) and observing whether a complementary fragment complex structure is formed in the detection system, thereby qualitatively or quantitatively determining whether the detection object exists in the sample to be detected.
In another preferred embodiment, said observing whether a complementary fragment complex is formed in said detection system comprises detecting a detectable signal generated by said complementary fragment complex.
In another preferred embodiment, in said detection system, a substrate is present which is catalysed by said complementary fragment complex.
In another preferred embodiment, the method is an in vitro or in vivo method.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
In another preferred embodiment, the method is a diagnostic method.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows an active protein that can be split into two complementary fragments;
FIG. 2 shows a schematic representation of an example of the invention in which two complementary fragments are attached to two different NB elements, respectively.
FIG. 3 shows a schematic representation of another example of the invention, wherein two complementary fragments of a protein are linked to NB1 and NB2 elements, respectively; in addition, two complementary fragments of the protein were linked to NB3 and NB4 elements, respectively.
FIG. 4 shows a schematic representation of another example of the invention, wherein two complementary fragments are linked to two different tandem NB elements, namely tandem NB 1-NB 2, tandem NB3 and NB4, respectively.
FIG. 5 shows the assembly of two single domain antibodies acting on the RBD domain of the S protein of the novel coronavirus, linked to complementary fragments of luciferase, into a system for the detection of the novel coronavirus.
FIG. 6 shows the data for virus detection using the system for detecting new coronavirus shown in FIG. 5.
Detailed Description
The present inventors have conducted extensive and intensive studies and, as a result, have surprisingly found that single domain antibodies (including but not limited to nanobodies and artificially synthesized single domain antibodies, the definition of which is the same as that of the single domain antibodies mentioned herein and will not be described in detail) can be linked to complementary fragments of enzymes for the detection of proteins, peptides and nucleic acids of biomolecules, thereby completing the present invention.
Term(s) for
Principle of action
1. The scFV or Fab fragment of the single domain antibody or antibody acting on a different biological substance to be detected is linked to a complementary fragment of protease, and when the different single domain antibody or scFV or Fab is bound to an object to be detected, the complementary fragments of the enzyme (the complementary fragments of the enzyme means that the enzyme is divided into different fragments and the different fragments are combined together to restore the activity of the enzyme) are brought into proximity to form an enzyme having complementary activity, and the presence or absence of a biomolecule such as a protein to be detected, a nucleic acid, a virus, a pathogenic microorganism, or the like, and the content thereof are detected by catalyzing the substrate with the enzyme to detect the amount of the substrate. Wherein said different single domain antibodies or complementary fragments of antibodies of different species are linked in various ways, and wherein upon linkage the antibodies draw the complementary fragments together to form the active complementary enzyme.
Protein modules and protein elements that can be assembled with one another
Each protein element of the invention has the structure of formula I,
Pi－(Li－NBi)m(I)
for example, Pi may be a biological enzyme.
In the case of a biological enzyme, the complementary fragment of the biological enzyme is linked to the scFV or Fab fragment of a different single domain antibody or antibody, and may be linked to the scFV or Fab fragment of the single domain antibody or antibody at the N-terminus or C-terminus, and the linking region may be in any manner, such as a linking sequence of amino acids, or a nucleic acid sequence, a polymeric molecule, or the like.
Complementary fragments of biological enzymes are linked to different single domain antibodies or scFV or Fab fragments of antibodies, expressed recombinantly by means of protein fusion or separately and linked by chemical or biological methods.
The substrate for the enzyme to form an active enzyme upon linkage of a complementary fragment of a biological enzyme to a different single domain antibody or scFV or Fab fragment of an antibody is fluorescein, a modified DNA fragment, a modified polypeptide fragment, the product being a detectable fluorescent, electrical or chemical signal.
Active protein and reconstituted complementary fragment composite structure
In the present invention, the kind of active protein is not particularly limited. Representative biological enzymes that can be linked to a scFV or Fab fragment of a different single domain antibody or antibodies are enzymes that can be divided into complementary fragments, including (but not limited to): luciferase, nuclease, endonuclease, protease, and fluorescent protein.
Typically, complementary fragments of biological enzymes are linked to different single domain antibodies or scFV or Fab fragments of antibodies to form a detection system, including single use, several used simultaneously to increase detection sensitivity.
Complementary fragments of the biological enzyme are linked to different monovalent or bivalent or multivalent tandem single domain antibodies or scFV or Fab fragments of the antibodies to improve detection sensitivity.
Applications of
The present invention provides a new method for detecting biological substances, which is used for detecting proteins, peptides, nucleic acids, viruses, fungi, bacteria, chlamydia, etc., and is used for detecting diseases and insect pests in clinical diagnosis, livestock breeding industry and agricultural planting
The detection method of the invention utilizes the protein component of the invention, and the protein component is used for detecting biological substances such as protein, polypeptide, nucleic acid and the like, clinically detecting the content of biomarker molecules of diseases, diagnosing the diseases and being also used for routine physical examination items; used for detecting organisms such as viruses, bacteria, fungi and the like and diagnosing the infection sources of human, animals and plants; can also be used for laboratory research to detect the content and dynamic change of the biological molecules
The main advantages of the invention include:
(a) the protein module of the present invention can rapidly detect a sample without separating a detection substance.
(b) When the protein assembly is detected, the signal is amplified due to the substance generated by the catalysis of the enzyme, so that the detection sensitivity is high.
(c) When the protein assembly is used for detecting a sample, the operation steps are simple.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Example 1 detection of novel coronavirus
After the two single domain antibodies acting on the RBD domain on the S protein of the new coronavirus are connected with the complementary fragment of luciferase, a system for detecting the new coronavirus is assembled and used for quickly detecting the new coronavirus. As shown in figures five and six.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (6)

1. An mutually assemblable protein module, said protein module comprising: n protein elements, wherein n is 2, 3 or 4, each protein element having the structure of formula I,
Pi－(Li－NBi)m (I)
in the formula (I), the compound is shown in the specification,
i is a positive integer from 1 to n;
pi is the i' th complement of an active protein;
li is nothing, a bond or a linker;
NBi is the ith anchor element connected to Pi through Li;
m is 1 to 3;
wherein each Pi is a complementary fragment from the same active protein and each Nbi is independently targeted to and binds to a predetermined assay target;
wherein, when the test object is present, each protein element binds to the test object via its respective anchor element such that all Pi together form a reconstituted complementary fragment complex (i.e., a reconstituted active protein) that has or retains the activity of the active protein;
when the test object is not present, and the protein elements are not bound to the test object, all Pi do not together form the reconstructed complementary fragment complex (i.e., a complex structure with a function cannot be formed).
2. A nucleotide molecule encoding the mutually assemblable protein modules of claim 1.
3. An expression vector comprising the nucleotide molecule of claim 2.
4. A host cell comprising the expression vector of claim 3; or the host cell has integrated into its genome the nucleotide molecule of claim 2.
5. Use of the mutually assemblable protein modules of claim 1 in the preparation of a protein, peptide, nucleic acid, virus, bacteria, fungus, chlamydia, protist assay or kit.
6. A method of detection, comprising:
(a) providing a sample to be tested, wherein the sample may contain a predetermined test object;
(b) mixing said sample with said mutually assemblable protein moieties of claim 1 to form a detection system;
(c) and observing whether a complementary fragment complex structure is formed in the detection system, thereby qualitatively or quantitatively determining whether the detection object exists in the sample to be detected.
CN202110032085.1A 2021-01-11 2021-01-11 Assembled protein element and application thereof Pending CN112876568A (en)

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Cited By (1)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022148491A1 (en) * 2021-01-11 2022-07-14 上海勇鉴生物科技有限公司 Assembled protein element and use thereof

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