CN116120446A - Antibody protein composition of NOTCH2NLC gene encoded protein, detection reagent and kit - Google Patents

Abstract

The application provides an antibody protein composition, a detection reagent and a kit of NOTCH2NLC gene encoded protein, wherein the antibody protein comprises a first antibody protein and/or a second antibody protein, the amino acid sequence of the first antibody protein is shown as SEQ ID NO.1, and the amino acid sequence of the second antibody protein is shown as SEQ ID NO. 2. The antibody protein related by the invention adopts the antibody capable of specifically recognizing the protein encoded by the NIID disease pathogenic gene, can specifically recognize and combine with the pathogenic gene encoded protein, can detect the pathogenic protein in skin tissue or cerebrospinal fluid, saliva, blood, urine and other liquid samples derived from patients, effectively improves the key defects of poor specificity and low repeatability, and provides an accurate and effective technical scheme for clinical diagnosis.

Description

Antibody protein composition of NOTCH2NLC gene encoded protein, detection reagent and kit

Technical Field

The disclosure relates to the technical field of detection of NIID pathogenic proteins, in particular to an antibody protein composition, a detection reagent and a kit of a NOTCH2NLC gene encoded protein.

Background

Neuronal nuclear inclusion body disease (Neuronal Intranuclear Inclusion Disease, NIID) is a neurodegenerative disease characterized by the widespread presence of neuronal nuclear inclusion bodies. The clinical manifestations of NIID are complex and diverse, and are mainly manifested by muscle weakness and dementia, and in addition, parkinsonism, cerebellar ataxia, tremors, episodic disturbance of consciousness, sensory disturbance, dyskinesia, and the like can be seen. There are studies reporting that the NOTCH2NLC gene GGC repeats abnormally amplified as a pathogenic mutation of NIID.

At present, clinically, auxiliary diagnostic means of NIID comprise gene detection, skin biopsy, magnetic resonance detection and the like, wherein the gene detection result cannot reflect the progress of diseases, the relevance of the magnetic resonance result and clinical phenotype is not clear, and the skin biopsy is an important auxiliary diagnostic means at present. Skin biopsy is mainly carried out by taking skin tissue of a patient, preparing a tissue section, then carrying out HE staining, and forming inclusion bodies in nuclei under an optical microscope. However, similar endosomes exist in skin tissues of patients with neurodegenerative diseases having similar clinical phenotypes such as fragile X-associated tremor/ataxia syndrome (FXTAS), and thus skin biopsy diagnosis using HE staining has a disadvantage of poor specificity.

Meanwhile, with the development of molecular biology and diagnostic techniques, liquid biopsies are gradually in the brand-new corner. Liquid biopsy diagnoses diseases by sampling cerebrospinal fluid, saliva, hydrothorax, blood, ascites, urine and the like, and compared with tissue biopsy, the detection of pathogenic proteins in body fluid derived from a patient by applying specific antibodies has the advantages of noninvasive performance, strong repeatability, realization of early diagnosis and dynamic monitoring, however, no specific antibodies can develop related liquid biopsy at present.

Therefore, there is a need to develop a detection means specific to the NIID.

The information disclosed in the background section of this application is only for enhancement of understanding of the general background of this application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The application provides an antibody protein composition of a protein encoded by a NOTCH2NLC gene, which has the advantage of specifically recognizing NOTCH2NLC polyglycine (hereinafter called NOTCH2 NLC-polyG) and NOTCH2NLC polyalanine (hereinafter called NOTCH2 NLC-polyA) proteins.

In a first aspect, the present application provides an antibody protein composition of NOTCH2NLC gene encoded protein and its expression sequence, which can be used for the detection of NIID. The NOTCH2NLC gene coded antibody protein comprises a first antibody protein and/or a second antibody protein, wherein the first antibody protein is NOTCH2NLC-polyG antigen protein, and the amino acid sequence of the first antibody protein is shown as SEQ ID NO. 1; the amino acid sequence of the second antibody protein, namely NOTCH2NLC-polyA antigen protein, is shown as SEQ ID NO. 2.

In a second aspect, the present application provides a detection reagent comprising the aforementioned antibody protein.

A third aspect of the present application provides a kit comprising the aforementioned antibody protein.

Advantageous effects

Compared with the HE staining specificity of skin biopsy sections, the defect of the inclusion body in the neuron nucleus in diseases such as fragile X-related tremor/ataxia syndrome (FXTAS) cannot be effectively identified. The antibody protein composition of the NOTCH2NLC gene encoded protein can specifically identify and combine the protein encoded by the pathogenic gene by adopting an antibody specifically identifying the protein encoded by the NIID disease pathogenic gene, can detect pathogenic proteins in human skin tissues or liquid samples of cerebrospinal fluid, saliva, blood and urine, can be used for liquid biopsy, effectively overcomes the key defects of poor specificity and poor repeatable operability in the prior detection technology, and provides an accurate and effective technical scheme for clinical diagnosis.

Drawings

FIG. 1A shows that GFP aggregate formation by both NOTCH2NLC-polyG and NOTCH2NLC-polyA can be observed under a fluorescence microscope after transfection of the NOTCH2NLC- (GGC) 98 plasmid in cells;

FIG. 1B shows that the formation of NOTCH2NLC-ployG aggregates can be detected using GFP antibodies;

FIG. 1C shows that the formation of NOTCH2NLC-ployA aggregates can be detected using GFP antibodies;

FIG. 2A shows a partial protein sequence of NOTCH2NLC-polyG, the specific protein sequence shown in underlined: CAARPPRMH is the antigen recognition sequence of an antibody;

FIG. 2B shows a partial protein sequence of NOTCH2NLC-polyA, the specific protein sequence shown in underlined: EEAATEKMPALRRSAVG is the antigen recognition sequence of an antibody;

FIG. 2C shows a sequence alignment of the NOTCH2, NOTCH2NLC-polyA and NOTCH2NLC-polyB proteins, the yellow part of the sequence in the figure being the recognition sequence of the PEP122 antibody of the NOTCH2NLC-polyG protein.

FIG. 2D shows that the antigen recognition sequence of the NOTCH2NLC-polyA antibody was aligned with human and mouse proteins, and no highly homologous protein sequences were found.

FIG. 2E shows the results of the immune response of PEP122 serum and purified antibodies produced after injection of CAARPPRMH polypeptide into two experimental rabbits.

FIG. 2F shows the results of the immune response of PEP123 serum and purified antibodies produced after injection of EEAATEKMPALRRSAVG polypeptide into two experimental rabbits.

FIG. 3A shows that the result of cellular immunofluorescence shows that the PEP122 antibody-labeled polyG aggregate and the flag tag protein expressed by fusion of the NOTCH2NLC-polyG protein have good co-localization;

FIG. 3B shows that the cell immunofluorescence results show that the PEP122 antibody-labeled polyA aggregate and the flag tag protein expressed by the fusion of the NOTCH2NLC-polyA protein have good co-localization;

FIG. 3C shows Western blot results demonstrating that PEP122 antibody specifically recognizes poly G protein, the size of the protein band is consistent with the results obtained when GFP tag protein was used in FIG. 1B;

FIG. 3D shows Western blot results demonstrating that PEP123 antibodies specifically recognize polyA protein, the size of the protein bands being consistent with the results obtained when GFP tag protein was used in FIG. 1C;

FIG. 4A shows immunohistochemical staining of FXTAS and NIID patient skin sections using PEP122 antibody, demonstrating that PEP122 is able to specifically recognize NOTCH2NLC-polyG aggregate A, indicated by the red arrow, but not FXTAS-polyG protein, in NIID patient skin;

FIG. 4B immunohistochemical staining of FXTAS and NIID patient skin sections with PEP123 antibody demonstrated that PEP123 was able to specifically recognize NOTCH2NLC-polyA aggregate B, indicated by the red arrow, but not FXTAS-polyA protein in NIID patient skin.

Detailed Description

For the purposes of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present disclosure, but not all embodiments. Based on the embodiments in this disclosure, all other embodiments that a person of ordinary skill in the art would obtain without making any inventive effort are within the scope of protection of this disclosure.

The technical scheme of the present disclosure is described in detail below with specific examples. The following embodiments may be combined with each other, and some embodiments may not be repeated for the same or similar concepts or processes.

The following are partial terms abbreviated or abbreviated corresponding full spellings or full names:

neuronal nuclear inclusion body disease, abbreviated NIID;

fragile X-associated tremor/ataxia syndrome, abbreviated FXTAS;

NOTCH2NLC polyglycine, called NOTCH2NLC-polyG for short;

NOTCH2NLC polyalanine, NOTCH2NLC-polyA;

green fluorescent protein Green fluorescent protein, GFP for short.

The existing pathogenesis research shows that a NOTCH2NLC-polyG protein aggregate generated by repeated expansion coding of a NOTCH2NLC gene GGC is one of important components of an endosome, so that the antibody which specifically recognizes the NOTCH2NLC-polyG protein aggregate is used for detecting a sample to be detected, such as skin slices, peripheral blood or cerebrospinal fluid, and the like, and has good application prospect.

1. Protein possibly produced by NOTCH2NLC after clear mutation

The inventor finds that repeated expansion of the NOTCH2NLC gene GGC can code and generate proteins such as NOTCH2NLC-polyG, NOTCH2NLC-polyA and the like, and the evidence is as follows:

(1) As shown in FIG. 1A, after transfection of the NOTCH2NLC- (GGC) 98-plasmid carrying 98 GGC repeats with pathogenicity in HEK293 cells, protein aggregate formation of large amounts of NOTCH2NLC-polyG-GFP aggregates and small amounts of NOTCH2NLC-polyA-GFP was observed under a fluorescence microscope; from the enlarged view, it can be seen that NOTCH2NLC-poly g aggregates are present in the nucleus and peri-nucleus, whereas NOTCH2NLC-poly a aggregates are present in the nucleus.

(2) As shown in fig. 1B and 1C, NOTCH2NLC plasmids of 17 GGC repeats (normal control) and 98 GGC repeats (mutant) were transfected in HEK293 cells, respectively, and expression of NOTCH2 NLC-protein was detected in Western blot experiments using GFP antibodies after 48 hours. The discovery is as follows:

as shown in FIG. 1B, the normal NOTCH2NLC-polyG (17) and NOTCH2NLC-polyA (17) proteins have a molecular weight around 35KD, while the mutated NOTCH2NLC-polyG (98) and NOTCH2NLC-polyA (98) proteins are greater than 250KD, suggesting the formation of aggregates.

The above experimental results indicate that NOTCH2NLC encoding with 98 GGC repeats produces NOTCH2NLC-poly g (98) and NOTCH2NLC-poly a (98) protein aggregates.

2. Design and preparation of antibodies specifically recognizing NOTCH2NLC protein products

To prepare specific antibodies for detection of the above proteins, we first analyzed to find specific sequences of NOTCH2NLC-poly g and NOTCH2NLC-poly a proteins as antigen recognition sequences of the antibodies.

Protein sequences of NOTCH2NLC-poly g and NOTCH2NLC-poly a are shown in fig. 2A and 2B, with the ellipses representing amplified poly g or poly a proteins, and the underlined amino acid sequences being the antigen recognition sequence (designated PEP 122) recognizing NOTCH2NLC-poly g and the antigen recognition sequence (designated PEP 123) recognizing NOTCH2NLC-poly a, respectively. To further verify the specificity of PEP122, we aligned the NOTCH2NLC-poly g protein with the NOTCH2, NOTCH2NLC-poly a and NOTCH2NLC-poly b proteins of the same family, as shown in fig. 2C, according to the NOTCH2NLC codon analysis, it was found that most of the three proteins were very highly homologous in sequence, while the underlined sequences had very good specificity, and their physicochemical properties met the requirements of antibody production. Therefore, this suggests that the protein sequence can be used as an antigen recognition sequence for the poly G antibody PEP 122.

For NOTCH2NLC-polyA, since NOTCH2NLC-polyA is not a protein generated by classical ATG codon initiation translation, peptide sequences with physicochemical properties meeting the requirements of antibody preparation in polyA proteins are selected, and in BLAST protein comparison tools of BCBI websites, polyA antigen recognition sequences are compared with all protein sequences of human and mice to verify the specificity. As shown in FIG. 2D, it was confirmed that no protein highly homologous to EEAATEKMPALRRSAVG sequence was found, suggesting that this sequence could be used as an antigen recognition sequence for the polyA antibody PEP 123.

The inventors selected the antigen recognition sequence of the polyA antibody described above: EEAATEKMPALRRSAVG; antigen recognition sequence of poly g antibody: CAARPPRM; the two antigen recognition sequences are respectively synthesized into polypeptides, injected into two rabbits for multiple immune reactions, and finally two antibodies of PEP122 antibody aiming at NOTCH2NLC-polyG protein and PEP123 antibody aiming at NOTCH2NLC-polyA protein are obtained.

As shown in FIGS. 2E-2F, the ELISA method is adopted to detect the immune reaction of serum and purified antibodies generated after 2 rabbits are respectively injected with PEP122 and PEP123 polypeptides, and the results show that in the section staining experiments after three PEP122 injections of experimental rabbits 1# and 2# and after three PEP123 injections of experimental rabbits 3# and 4#, the results of the first injection and the third injection are blue, and a good immune staining reaction occurs, so that the antibody protein of the poly-G antigen selected by the application can specifically identify NOTCH2NLC-polyG protein, and the antibody protein of the poly-A antigen can specifically identify NOTCH2NLC-polyA protein.

3. Validation of antibody availability and specificity

The applicant then further tested the validity and specificity of PEP122 antibodies and PEP123 antibodies using a cell model.

As shown in FIG. 3A, after transfection of the NOTCH2NLC- (GGC) 98 plasmid carrying 98 GGC repeats with pathogenicity in HEK293 cells, the results of cellular immunofluorescence showed that the NOTCH2NLC-polyG protein detected with the flag antibody (as shown in the left panel of FIG. 3A) had a good co-localization with the NOTCH2NLC-polyG aggregates detected with the PEP122 antibody (as shown in the middle panel of FIG. 3A), as shown in the right panel of FIG. 3A.

As shown in FIG. 3B, after transfection of the NOTCH2NLC- (GGC) 98 plasmid carrying 98 GGC repeats with pathogenicity in HEK293 cells, the results of cellular immunofluorescence showed that the NOTCH2NLC-polyA protein detected with the HA antibody (as shown in the left panel of FIG. 3B) had good co-localization with the NOTCH2NLC-polyA aggregates detected with the PEP123 antibody (as shown in the middle panel of FIG. 3B), as shown in the right panel of FIG. 3B.

As shown in FIGS. 3C and 3D, after transfection of the NOTCH2NLC- (GGC) plasmid in HEK293 cells, the PEP122 antibody specifically recognized a NOTCH2NLC-polyG protein aggregate with a molecular weight of more than 250KD in Western blot experiments, and the PEP123 antibody specifically recognized a NOTCH2NLC-polyA protein aggregate with a molecular weight of more than 250KD, which protein band sizes were consistent with those obtained when using the tag proteins in FIGS. 1B and 1C. The experimental results show that the PEP122 antibody can well identify the NOTCH2NLC-polyG protein, and the PEP123 antibody can well identify the NOTCH2NLC-polyA protein. The effectiveness of the antibodies of the present application was confirmed.

4. Patient skin tissue detection NOTCH2NLC protein

As described in the background section, staining of skin tissue using HE staining does not distinguish NIID from FXTAS, because FXTAS is a disease caused by repeated aberrant amplification of the FMR1 gene GGC, a mutation in the gene that encodes the production of FMR1-polyG and FMR1-polyA protein aggregates.

To further verify the effectiveness and specificity of the antibodies prepared by the inventors. Next we performed immunohistochemical staining of FXTAS and skin biopsies of NIID patients using PEP122 and PEP123 antibodies, as indicated by the arrows in fig. 4A, PEP122 antibodies were able to detect the presence of aggregates in nuclei in NIID patients but not in FXTAS patient skin groups, indicating that PEP122 was able to specifically recognize NOTCH2NLC-poly g aggregates in NIID patient skin but not FMR1-poly g protein aggregates.

As shown by the arrow in fig. 4B, PEP123 antibodies were able to detect the presence of aggregates in the nuclei in the skin group of NIID patients but not FXTAS patients, suggesting that PEP123 antibodies were able to specifically recognize NOTCH2NLC-polyA aggregates in the skin of NIID patients, but not FMR1-polyA protein aggregates.

The above experimental results show that PEP122 and PEP123 antibodies are effective and specific for recognizing NOTCH2NLC-poly g aggregates and NOTCH2NLC-poly a aggregates, respectively, in the skin group of NIID patients.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present disclosure, and not for limiting the same; although the present disclosure has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present disclosure.

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Claims (3)

1. An antibody protein composition of NOTCH2NLC gene coded protein comprises a first antibody protein and/or a second antibody protein, wherein the amino acid sequence of the first antibody protein is shown as SEQ ID NO.1, and the amino acid sequence of the second antibody protein is shown as SEQ ID NO. 2.

2. A detection reagent comprising the antibody protein composition of the NOTCH2NLC gene encoded protein of claim 1.

3. A kit comprising an antibody protein composition of the NOTCH2NLC gene encoded protein of claim 1.

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