Recombinant protein for detecting paraneoplastic Yo antibody by CBA method and application thereof

Abstract

The application discloses a recombinant protein for detecting paraneoplastic Yo antibody by CBA method and application, and belongs to the technical field of biological medicine and engineering. The amino acid sequence of the recombinant protein comprises, in sequence, a secretion signal peptide, a CDR2 protein partial sequence, a CDR2L protein partial sequence, a transmembrane region and a fluorescent label. The secretion signal peptide is a human albumin signal peptide ALB. The transmembrane region is CD8a hinge. The fluorescent label is mCherry. The application constructs a novel recombinant protein which can be stably overexpressed on the cell membrane of eukaryotic cells by intercepting specific partial sequences of CDR2 protein and CDR2L protein, and redesigning and fusing a fluorescent label by using a secretion signal peptide, a transmembrane sequence and a connecting peptide. The recombinant protein retains the core antigen regions of the two, can effectively overcome the insufficiency in sensitivity and specificity, can significantly improve the detection rate of paraneoplastic Yo antibody when CBA method is used for detection, and can reduce the false positive rate, so that the requirement of clinical detection can be better met.

Description

TECHNICAL FIELD

[0001] The present application is a recombinant protein for detecting paraneoplastic Yo antibody by CBA method and application, and particularly relates to a recombinant protein for detecting paraneoplastic syndrome Yo antibody based on cell immunofluorescence method (Cell Based Assay, CBA method) and related application thereof, and belongs to the technical field of biological medicine engineering. BACKGROUND

[0002] Paraneoplastic neurological syndromes (PNS) are neurological syndromes associated with cancer. Recent studies have shown that the occurrence of many PNS is closely related to antibodies against tumor-expressed neural proteins (neural antibodies), which suggests that the pathogenesis of most PNS is immune-mediated. These immune responses can involve the nervous system, skin, endocrine system and other systems, thereby causing a variety of clinical symptoms. Antibody detection plays a crucial role in the diagnosis of PNS. Currently, common paraneoplastic-related antibodies include Hu, Yo, CV2, Ri, Ma2, SOX1, Amphiphysin, etc. These antibodies have been widely used in clinical detection to assist in the diagnosis of PNS. Detection of neural antibodies is of great value in suggesting the presence of potential tumors and their types, and in diagnosing neurological syndromes as paraneoplastic syndromes, and has been included in the international expert group paraneoplastic neurological syndrome diagnostic criteria (2021, Updated Diagnostic Criteria for Paraneoplastic Neurologic Syndromes).

[0003] Paraneoplastic cerebellar degeneration (PCD) is the most common type of PNS, which is closely related to ovarian cancer and breast cancer. In PCD patients, the expression of neural antigens in related ovarian cancer is common and is a prerequisite for triggering immune response. Yo antibody is the main neural antibody in the serum and cerebrospinal fluid of PCD patients and ovarian cancer patients, therefore, detection of Yo antibody is of great significance for the auxiliary diagnosis of PNS and PCD.

[0004] In the field of Yo antibody research, it is currently believed that there are three cerebellum antigens associated with Yo antibodies, which are Cerebellar Degeneration-Related Protein 2 (CDR2), CDR2-like (CDR2L), and Cerebellar Degeneration-Related Protein 1 (CDR1). CDR2L is named as CDR2-like because its sequence has about 45% sequence identity with CDR2. Early studies (1987, Cloning of a brain protein identified by autoantibodies from a patient with paraneoplastic cerebellar degeneration) suggested that CDR2 protein is the only Yo antigen expressed in ovarian cancer and is also the main target of Yo antibodies in Purkinje cells of the cerebellum. Based on this, the detection of Yo antibodies is usually carried out by Western blotting using recombinant CDR2 protein.

[0005] However, recent studies have found that serum from patients with paraneoplastic cerebellar degeneration (PCD) reacts to both CDR2 and CDR2L proteins, and the pathogenic role of CDR2L antibodies in PCD has been confirmed (2013, CDR2L antibodies: a new player in paraneoplastic cerebellar degeneration / CDR2L antibodies: a new player in paraneoplastic cerebellar degeneration). In addition, some researchers have conducted clinical comparative analysis on the results of using only immunoblotting to detect Yo antibodies, and the results show that there are more false positives when only using immunoblotting to detect anti-CDR2 (2017, Cerebellar degeneration-related proteins 2 and 2-like are present in ovarian cancer in patients with and without Yo antibodies; 2023, A cerebellar degeneration-related protein 2-like cell-based assay for anti-Yo detection in patients with paraneoplastic cerebellar degeneration / CDR2 protein and CDR2L protein are present in ovarian cancer patients with and without Yo antibodies; 2023, A cerebellar degeneration-related protein 2-like cell-based assay for anti-Yo detection in patients with paraneoplastic cerebellar degeneration). Therefore, the detection method of Yo antibodies relying only on the immunoblotting of CDR2 recombinant protein is likely to miss detection and is no longer suitable for clinical detection.

[0006] Studies have also been conducted on CDR2L as an anti-Yo antibody detection. Studies have found that using a cell matrix adsorption method (Cell-Based Assay, CBA) expressing the full length of CDR2L can improve the specificity of Yo antibody detection and reduce false positives caused by using only CDR2 recombinant protein immunoblotting (2023, A cerebellar degeneration-related protein 2-like cell-based assay for anti-Yo detection in patients with paraneoplastic cerebellar degeneration). However, studies have also pointed out that the combined use of CDR2 and CDR2L can obtain the most reliable detection results, and whether a single anti-CDR2L positive detection can be used as a sufficient biomarker for Yo-PCD diagnosis still needs to be further evaluated. In addition, since CDR2L is expressed in the cytoplasm, the use of fixed and permeabilized CBA detection often results in high background signal of fixed and permeabilized CBA detection in patient serum, thereby reducing the detection sensitivity.

[0007] In summary, accurate detection of Yo antibodies against CDR2 and CDR2L proteins is crucial for the diagnosis of paraneoplastic neurological syndrome (PNS) and paraneoplastic cerebellar degeneration (PCD). However, the existing detection methods, whether based on CDR2 protein immunoblotting or combined detection of CDR2 and CDR2L, have deficiencies in sensitivity and specificity in clinical application. Therefore, there is an urgent need to develop a new detection technology that can simultaneously target Yo antibodies against CDR2 and CDR2L and has higher sensitivity and specificity. SUMMARY

[0008] The purpose of the present application is to provide a recombinant protein for CBA detection of paraneoplastic Yo antibodies and its application. The recombinant protein is constructed by fusing specific segments of CDR2 protein and CDR2L protein, and retains the core antigen regions of each of them. Compared with the existing CDR2 protein-based immunoblotting or combined detection of CDR2 and CDR2L, the present application can effectively overcome the deficiencies in sensitivity and specificity, significantly improve the detection rate of paraneoplastic Yo antibodies and reduce the false positive rate when using CBA for detection, thereby better meeting the needs of clinical detection.

[0009] The application is realized by the following technical scheme: a recombinant protein for detecting paraneoplastic Yo antibody by CBA method (live cell immunofluorescence method), the amino acid sequence of the recombinant protein comprises in sequence: a secretion signal peptide, a CDR2 protein partial sequence as shown in SEQ ID NO: 1, a CDR2L protein partial sequence as shown in SEQ ID NO: 2, a transmembrane region and a fluorescent label,

[0010] The secretion signal peptide is human albumin signal peptide ALB.

[0011] The transmembrane region is CD8a hinge.

[0012] The fluorescent label is mCherry.

[0013] Preferably, the amino acid sequence of the secretion signal peptide is shown in SEQ ID NO: 3.

[0014] Preferably, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 4.

[0015] Preferably, the amino acid sequence of the fluorescent label is shown in SEQ ID NO: 5.

[0016] Preferably, the amino acid sequence of the recombinant protein further comprises a connecting peptide A connecting the CDR2L protein partial sequence and the transmembrane region, and a connecting peptide B connecting the transmembrane region and the fluorescent label.

[0017] Preferably, the amino acid sequence of the connecting peptide A is shown in SEQ ID NO: 6; and the amino acid sequence of the connecting peptide B is shown in SEQ ID NO: 7.

[0018] The application further provides specific applications of the above-mentioned recombinant protein, including:

[0019] (I) the application of the recombinant protein in the preparation of a product for detecting paraneoplastic Yo antibody, characterized in that: the stable transfection cell obtained by transfecting a host cell with a nucleic acid encoding the recombinant protein to express is used as a detection product, and the detection of paraneoplastic Yo antibody is carried out by CBA method (cell immunofluorescence method).

[0020] The host cell includes CHO cell or HEK293 cell.

[0021] (II) a CBA method (cell immunofluorescence method) reagent for detecting paraneoplastic Yo antibody, the reagent comprising a transfection cell stably expressing the recombinant protein.

[0022] (III) a CBA method (cell immunofluorescence method) kit for detecting paraneoplastic Yo antibody, the kit comprising the above-mentioned reagent.

[0023] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0024] (1) The present application constructs a novel recombinant protein that can be stably overexpressed on the cell membrane of eukaryotic cells by intercepting specific partial sequences of CDR2 protein and CDR2L protein, and redesigning and fusing a fluorescent tag using a secretion signal peptide, a transmembrane sequence and a linker peptide. The recombinant protein retains the key antigen epitopes of CDR2 and CDR2L, providing a structural basis for high-sensitivity and high-specificity detection.

[0025] (2) Compared with traditional CDR2 immunoblotting or combined detection of CDR2 and CDR2L, the detection system based on CBA method (cellular immunofluorescence method) of the present application significantly improves the detection rate, effectively reduces false positive results, and significantly improves the overall detection sensitivity and specificity.

[0026] (3) The novel recombinant protein constructed by the present application does not require co-transfection of multiple antigen genes, is easy to obtain a stable monoclonal cell line, greatly simplifies the establishment process of the cell model, is conducive to realizing standardized and large-scale production, and meets the actual application requirements of clinical detection.

[0027] (4) After a large number of clinical samples are verified, the present application shows excellent detection performance in the detection of Yo antibody related to paraneoplastic syndrome, and can better meet the requirements of accuracy and reliability of current clinical detection. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a schematic diagram of the three-dimensional conformation of CDR2 natural full-length protein.

[0029] Figure 2 is a schematic diagram of the three-dimensional conformation of CDR2L natural full-length protein.

[0030] Figure 3 is a schematic diagram of the three-dimensional conformation of the recombinant protein of the present application.

[0031] Figure 4 is a schematic diagram of the structure comparison of the three-dimensional conformation of the recombinant protein of the present application and the natural proteins of CDR2 and CDR2L.

[0032] Figure 5 is a lentiviral vector diagram of the recombinant protein of the present application.

[0033] Figure 6 is a cellular immunofluorescence diagram of a positive sample in Example 3.

[0034] Figure 7 is a cellular immunofluorescence diagram of a negative sample in Example 3. DETAILED DESCRIPTION

[0035] The invention purposes, technical solutions and beneficial effects will be further described in detail below.

[0036] It should be pointed out that the following detailed description is exemplary and is intended to provide further description of the claimed invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the invention belongs.

[0037] The present application aims to provide a recombinant protein with CDR2 and CDR2L partial sequence fusion, which can be stably overexpressed in the cytoplasm of eukaryotic cells and is extremely easy to obtain a stable cell line stably expressing antigen conformation. Specifically, the amino acid sequence of the recombinant protein comprises, in order: a secretion signal peptide, a CDR2 protein partial sequence, a CDR2L protein partial sequence, a linker peptide A, a transmembrane region, a linker peptide B and a fluorescent tag. Among them:

[0038] The secretion signal peptide is the human albumin signal peptide ALB, see SEQ ID NO: 3, and its amino acid sequence is:

[0039] MKWVTFISLLFLFSSAYS;

[0040] The CDR2 protein partial sequence is from the 43-158 amino acid sequence of the transcript isoform NM_001802.2, see SEQ ID NO: 1, and its amino acid sequence is:

[0041] TELEDSVQQMYTTNQEQLQEIEYLTKQVELLRQMNEQHAKVYEQLDVTARELEETNQKLVADSKASQQKILSLTETIECLQTNIDHLQSQVEELKSSGQGRRSPGKCDQEKPAPSF;

[0042] The CDR2L partial sequence is from the 159-465 amino acid sequence of the transcript isoform NM_014603.3, see SEQ ID NO: 2, and its amino acid sequence is:

[0043] PCLKELCTSP RCKDAFRLH S SLELGPRPLE QENERLQTL VGALRSQVSQ ERQRKERAER EYTAVLQEYSELERQLCEME ACRLRVQELE AELLELQQMK QAKTYLLGPDDHLAEALLAPLTQ APEADDPQPGRGDDLGAQDGVSSPAASPGHVVRKSCSDTALNAIVAKDPASRHAGNLTLHANSVRKRGMSILREVDEQYHALLEKYEELLSKCRQHGAGVRHAGVQTSRPISRDSSWRDLRGGEEGQGEVKAGEKSLSQHVEAVDKRLEQSQPEYKALFKEIFSRIQKTKADINATKVKTHSSK;

[0044] The amino acid sequence of the linker peptide A is shown in SEQ ID NO: 6:

[0045] GSTGGGGSGGGGSGGGGSGAASR;

[0046] The transmembrane region is the hinge of CD8a, i.e. the 183-206 amino acid sequence of CD8a hinge region NM_001768, shown in SEQ ID NO: 4:

[0047] IYIWAPLAGTCGVLLLSLVITLYC.

[0048] The amino acid sequence of the linker peptide B is shown in SEQ ID NO: 7:

[0049] ASGGGGSGGGGSSG;

[0050] The fluorescent tag is mCherry, shown in SEQ ID NO: 5:

[0051] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0052] Further, the recombinant protein (secretion signal peptide-CDR2 partial sequence-CDR2L partial sequence-linker A-transmembrane sequence-linker B-mCherry) obtained by the above amino acid sequence fusion construction, see SEQ ID NO: 8, the amino acid sequence is:

[0053] MKWVTFISLLFLFSSAYSTELEDSVQQMYTTNQEQLQEIEYLTKQVELLRQMNEQHAKVYEQLDVTARELEETNQKLVADSKASQQKILSLTETIECLQTNIDHLQSQVEELKSSGQGRRSPGKCDQEKPAPSFPCLKELCTSPRCKDAFRLHSSSLELGPRPLEQENERLQTLVGALRSQVSQERQRKERAEREYTAVLQEYSELERQLCEMEACRLRVQELEAELLELQQMKQAKTYLLGPDDHLAEALLAPLTQAPEADDPQPGRGDDLGAQDGVSSPAASPGHVVRKSCSDTALNAIVAKDPASRHAGNLTLHANSVRKRGMSILREVDEQYHALLEKYEELLSKCRQHGAGVRHAGVQTSRPISRDSSWRDLRGGEEGQGEVKAGEKSLSQHVEAVDKRLEQSQPEYKALFKEIFSRIQKTKADINATKVKTHSSKGSTGGGGSGGGGSGGGGSGAASRIYIWAPLAGTCGVLLLSLVITLYCASGGGGSGGGGSSGMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0054] To verify whether the antigen conformation of the constructed recombinant protein can be stably expressed, the Alpha Fold 3 software is used to predict the three-dimensional structure of CDR2 and CDR2L natural full-length protein clusters and the constructed recombinant protein, respectively, see Figures 1 to 3 wherein, Figure 1represents CDR2 protein, Figure 2 represents CDR2L protein, Figure 3 represents recombinant protein. See Figure 4 The structure alignment result shows that the three-dimensional structure of the recombinant protein is well overlapped with the three-dimensional structure of the antigen part of the natural proteins CDR2 and CDR2L, and both can normally expose the antigen binding site. This result shows that the recombinant protein successfully retains the key spatial conformation of the natural protein, and establishes a reliable structural basis for its use in the CBA method (cellular immunofluorescence method) for paraneoplastic YO antibody detection.

[0055] The application will be further described in detail below in conjunction with examples, but the embodiments of the application are not limited thereto.

[0056] Example 1: Construction of lentiviral vector

[0057] The above recombinant protein (secretion signal peptide-CDR2 partial sequence-CDR2L partial sequence-linker peptide A-transmembrane sequence-linker peptide B-mCherry) designed by the application is cloned into a lentiviral transfer plasmid to construct a lentiviral vector capable of stably expressing CDR2 and CDR2L recombinant proteins, and the map of the lentiviral vector constructed is shown in Figure 5 .

[0058] Example 2: Preparation of detection reagent for paraneoplastic Yo antibody

[0059] The lentiviral vector prepared in Example 1 is transfected into CHO or HEK293 cells, and then the cells are screened by the limiting dilution method to obtain monoclonal cells stably expressing the recombinant protein. The cells are treated with trypsin-EDTA solution to form a single cell suspension, and after cell counting, the cells are diluted to 1 cell / 100 µL by the limiting dilution method, and then inoculated into 96-well plates at 100 µL / well. The wells of single cell groups are screened, and the wells with strong red fluorescence are selected for freezing, primary antibody immunofluorescence detection. The cells that meet the requirements are used to prepare the detection reagent or kit for subsequent immunofluorescence experiments.

[0060] Inoculation of cell sheets: the de-enzymed recombinant protein monoclonal cells and control empty cells are mixed uniformly at a ratio of 1:2 of monoclonal cells to control cells, and inoculated into 96-well plates or cell climbing sheets at a total density of 2×10 5 / mL. F12K+10%FBS+NEAA is used for culture, and the culture is stopped when the confluence is 90-100% after about 48 hours of growth.

[0061] CBA method is used, and the specific operation is as follows: the stably transfected monoclonal cells and empty control cells selected are mixed and inoculated into 96-well cell culture plates, and after 48 hours of culture, they are directly used for cellular immunofluorescence detection of paraneoplastic Yo antibody.

[0062] Example 3: CBA detection method of paraneoplastic Yo antibody

[0063] The detection reagent of paraneoplastic Yo antibody prepared according to the method of Example 1 and Example 2 was subjected to CBA operation:

[0064] Prepare the serum of a patient clinically diagnosed as Yo antibody positive paraneoplastic syndrome / normal human serum as a sample, and the sample diluent contains the following components:

[0065] DMEM base medium, BSA (1%);

[0066] The sample was subjected to initial determination dilution with the sample diluent, and the serum dilution ratio was 1:10;

[0067] Sample incubation: incubate in a 37°C carbon dioxide incubator for 1 hour, and wash with PBS for 3 times;

[0068] Dilute the secondary antibody with the sample diluent: use goat anti-human IgG (H+L) with a dilution ratio of 1:500, incubate at room temperature for 45 minutes, and wash with PBS for 3 times;

[0069] Judgment: judge the sample positive and negative under a microscope.

[0070] Comparative Example 1

[0071] This comparative example uses the CDR2 full sequence (NM_001802.2: 1-454aa) to express the detection of paraneoplastic Yo antibody. The CDR2 full sequence and the fluorescent tag mCherry are connected by a linker peptide to form a lentivirus vector, and the CDR2 full sequence-linker A-mCherry is constructed to express the detection of paraneoplastic Yo antibody.

[0072] Referring to SEQ ID NO: 9, the amino acid sequence of the CDR2 full sequence is:

[0073] MLAENLVEEFEMKEDEPWYDHQDLQQDLQLAAELGKTLLDRNTELEDSVQQMYTTNQEQLQEIEYLTKQVELLRQMNEQHAKVYEQLDVTARELEETNQKLVADSKASQQKILSLTETIECLQTNIDHLQSQVEELKSSGQGRRSPGKCDQEKPAPSFACLKELYDLRQHFVYDHVFAEKITSLQGQPSPDEEENEHLKKTVTMLQAQLSLERQKRVTMEEEYGLVLKENSELEQQLGATGAYRARALELEAEVAEMRQMLQSEHPFVNGVEKLVPDSLYVPFKEPSQSLLEEMFLTVPESHRKPLKRSSSETILSSLAGSDIVKGHEETCIRRAKAVKQRGISLLHEVDTQYSALKVKYEELLKKCQEEQDSLSHKAVQTSRAAAKDLTGVNAQSEPVASGWELASVNPEPVSSPTTPPEYKALFKEIFSCIKKTKQEIDEQRTKYRSLSSHS.

[0074] Linker A see SEQ ID NO: 6, mCherry see SEQ ID NO: 5.

[0075] By constructing a eukaryotic transient expression vector containing the above sequence, CHO and 293 cells were transfected with Lipofectamine™ 3000 transfection reagent for 48 hours. Due to the expression of the full sequence CDR2, the protein was located in the cytoplasm and nucleus, so 4% paraformaldehyde was used for fixation and Triton X-100 permeabilization. The pH value of the fixing agent was 7.0-7.6, the fixed cell culture plate was soaked for 20 minutes, then the cell slices were washed with phosphate buffer for 3 times, 5 minutes each time, then 0.3% Triton X-100 was added for 5 minutes, then the cell slices were washed with phosphate buffer for 3 times. After cleaning, blocking solution (phosphate buffer and 5% bovine serum albumin) was added to the cell culture plate well, and blocked at room temperature for 1 hour.

[0076] Comparative Example 2:

[0077] This comparative example is the process of using CDR2L full sequence (NM_014603.3: 1-465aa) to express and detect paraneoplastic Yo antibody. The CDR2L full sequence and the fluorescent tag mCherry are connected by a linker to form a lentiviral vector, and the CDR2L full sequence-linker A-mCherry is constructed to express and detect paraneoplastic Yo antibody.

[0078] The amino acid sequence of the CDR2L full sequence is, with reference to SEQ ID NO: 10:

[0079] MRRAAGMEDFSAEEEESWYDQQDLEQDLHLAAELGKTLLERNKELEGSLQQMYSTNEEQVQEIEYLTKQLDTLRHVNEQHAKVYEQLDLTARDLELTNHRLVLESKAAQQKIHGLTETIERLQAQVEELQAQVEQLRGLEQLRVLREKRERRRTIHTFPCLKELCTSPRCKDAFRLHSSSLELGPRPLEQENERLQTLVGALRSQVSQERQRKERAEREYTAVLQEYSELERQLCEMEACRLRVQELEAELLELQQMKQAKTYLLGPDDHLAEALLAPLTQAPEADDPQPGRGDDLGAQDGVSSPAASPGHVVRKSCSDTALNAIVAKDPASRHAGNLTLHANSVRKRGMSILREVDEQYHALLEKYEELLSKCRQHGAGVRHAGVQTSRPISRDSSWRDLRGGEEGQGEVKAGEKSLSQHVEAVDKRLEQSQPEYKALFKEIFSRIQKTKADINATKVKTHSSK.

[0080] Linker A is shown in SEQ ID NO: 6 and mCherry is shown in SEQ ID NO: 5.

[0081] By constructing a eukaryotic transient expression vector containing the above sequence, CHO and 293 cells were transfected by Lipofectamine™ 3000 transfection reagent for 48 hours. Due to the expression of the full sequence CDR2L, the protein was located in the cytoplasm, so the same fixation and permeabilization method as Comparative Example 1 was used to process the cell slices.

[0082] Comparative Example 3:

[0083] This comparative example is the process of co-expression of CDR2 full sequence (NM_001802.2: 1-454aa) and CDR2L full sequence (NM_014603.3: 1-465aa) to detect paraneoplastic Yo antibody.

[0084] The vectors constructed in Comparative Example 2 and Comparative Example 3 were transiently transfected into CHO and 293 cells at a ratio of 1:1 using Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, the proteins were localized in the nucleus and cytoplasm due to expression of the full sequence CDR2 and CDR2L, so the same fixation and permeabilization method as in Comparative Example 1 was used to process the cell slices.

[0085] Comparative Example 4:

[0086] This comparative example is a process of fusion expression for detecting paraneoplastic Yo antibody using a secretion signal peptide + CDR2 full sequence (NM_001802.2: 1-454aa) + CDR2L full sequence (NM_014603.3: 1-465aa) + connecting peptide A + transmembrane region + connecting peptide B + mCherry.

[0087] The CDR2 full sequence is shown in SEQ ID NO: 9, the CDR2L full sequence is shown in SEQ ID NO: 10, the secretion signal peptide is shown in SEQ ID NO: 3, the transmembrane region is shown in SEQ ID NO: 4, the connecting peptide A is shown in SEQ ID NO: 6, the connecting peptide B is shown in SEQ ID NO: 7, and the mCherry is shown in SEQ ID NO: 5.

[0088] A eukaryotic transient expression vector containing the above sequences was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, the same method as in Example 3 was used for detection.

[0089] Comparative Example 5:

[0090] This comparative example is a process of fusion expression for detecting paraneoplastic Yo antibody using a secretion signal peptide + CDR2 partial sequence (NM_001802.2: 43-158aa) + CDR2L full sequence (NM_014603.3: 1-465aa) + connecting peptide A + transmembrane sequence + connecting peptide B + mCherry.

[0091] The CDR2 partial sequence is shown in SEQ ID NO: 1, the CDR2L full sequence is shown in SEQ ID NO: 10, the secretion signal peptide is shown in SEQ ID NO: 3, the transmembrane region is shown in SEQ ID NO: 4, the connecting peptide A is shown in SEQ ID NO: 6, the connecting peptide B is shown in SEQ ID NO: 7, and the mCherry is shown in SEQ ID NO: 5.

[0092] The eukaryotic transient expression vector containing the above sequence was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, cultured for 48 hours, and then detected by the same method as in Example 3.

[0093] Comparative Example 6:

[0094] This comparative example is the process of fusion expression of detecting paraneoplastic Yo antibody by using secretory signal peptide + CDR2 full sequence (NM_001802.2: 1-454aa) + CDR2L partial sequence (NM_014603.3: 159-465aa) + connecting peptide A + transmembrane sequence + connecting peptide B + mCherry.

[0095] The CDR2 full sequence is shown in SEQ ID NO: 9, the CDR2L partial sequence is shown in SEQ ID NO: 2, the secretory signal peptide is shown in SEQ ID NO: 3, the transmembrane region is shown in SEQ ID NO: 4, the connecting peptide A is shown in SEQ ID NO: 6, the connecting peptide B is shown in SEQ ID NO: 7, and the mCherry is shown in SEQ ID NO: 5.

[0096] The eukaryotic transient expression vector containing the above sequence was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, cultured for 48 hours, and then detected by the same method as in Example 3.

[0097] Comparative Example 7:

[0098] This comparative example is the process of fusion expression of detecting paraneoplastic Yo antibody by using secretory signal peptide + CDR2 partial sequence (NM_001802.2: 1-158aa) + CDR2L partial sequence (NM_014603.3: 159-465aa) + connecting peptide A + transmembrane sequence + connecting peptide B + mCherry.

[0099] The amino acid sequence of the CDR2 partial sequence (1-158aa) is shown in SEQ ID NO: 11.

[0100] MLAENLVEEFEMKEDEPWYDHQDLQQDLQLAAELGKTLLDRNTELEDSVQQMYTTNQEQLQEIEYLTKQVELLRQMNEQHAKVYEQLDVTARELEETNQKLVADSKASQQKILSLTETIECLQTNIDHLQSQVEELKSSGQGRRSPGKCDQEKPAPSF.

[0101] The CDR2L partial sequence refers to SEQ ID NO: 2, the secretion signal peptide refers to SEQ ID NO: 3, the transmembrane region refers to SEQ ID NO: 4, the connecting peptide A refers to SEQ ID NO: 6, the connecting peptide B refers to SEQ ID NO: 7, and the mCherry refers to SEQ ID NO: 5.

[0102] CHO and 293 cells were transfected by constructing a eukaryotic transient expression vector containing the above sequence, using Lipofectamine™ 3000 transfection reagent, and cultured for 48 hours, and then detected by the same method as in Example 3.

[0103] Comparative Example 8:

[0104] This comparative example is the process of fusion expression and detection of paraneoplastic Yo antibody using a secretion signal peptide + CDR2 partial sequence (NM_001802.2: 43-158aa) + CDR2L partial sequence (NM_014603.3: 159-435aa) + connecting peptide A + transmembrane sequence + connecting peptide B + mCherry.

[0105] The amino acid sequence of the CDR2L partial sequence (159-435aa) is shown in SEQ ID NO: 12.

[0106] PCLKELCTSPRCKDAFRLHSSSLELGPRPLEQENERLQTLVGALRSQVSQERQRKERAEREYTAVLQEYSELERQLCEMEACRLRVQELEAELLELQQMKQAKTYLLGPDDHLAEALLAPLTQAPEADDPQPGRGDDLGAQDGVSSPAASPGHVVRKSCSDTALNAIVAKDPASRHAGNLTLHANSVRKRGMSILREVDEQYHALLEKYEELLSKCRQHGAGVRHAGVQTSRPISRDSSWRDLRGGEEGQGEVKAGEKSLSQHVEAVDKRLEQSQPE.

[0107] The CDR2L partial sequence refers to SEQ ID NO: 2, the secretion signal peptide refers to SEQ ID NO: 3, the transmembrane region refers to SEQ ID NO: 4, the connecting peptide A refers to SEQ ID NO: 6, the connecting peptide B refers to SEQ ID NO: 7, and the mCherry refers to SEQ ID NO: 5.

[0108] The eukaryotic transient expression vector containing the above sequence was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, cultured for 48 hours, and then detected by the same method as in Example 3.

[0109] The fixed cells prepared in the above Example 3, Comparative Example 1 to Comparative Example 8 were added to samples clinically diagnosed as Yo antibody positive paraneoplastic neurologic syndrome. According to the new diagnostic criteria for paraneoplastic neurologic syndromes of international experts group (Updated Diagnostic Criteria for Paraneoplastic Neurologic Syndromes, 2021), 86 serum samples of patients with confirmed anti-Yo paraneoplastic neurologic syndrome were selected. These samples were all positive for Yo antibody by blotting and met the following three conditions: A. Can affect any part of the nervous system, often showing stereotyped clinical manifestations; B. Related to malignant tumors; C. Neurological diseases with immune-mediated pathogenesis.

[0110] The serum was diluted at a ratio of 1:10, 1:32, 1:100, and 1:320, incubated (incubated at 37°C for 1 hour), washed with PBS for 3 times, added with secondary antibody (goat anti-human IgG, diluted at a ratio of 1:500) and incubated (at room temperature for 45 minutes), after washing with PBS for 3 times, a small amount of PBS was added to soak the cells, and the results were observed under a microscope.

[0111] The results are shown in Figure 6 and Table 1 below: Comparison of Yo antibody positive paraneoplastic neurologic syndrome antibody detection table.

[0112] Figure 6 The cell immunofluorescence images of the positive samples in Example 3 are shown in the figure, where A is the red fluorescence of the CDR2 and CDR2L fusion protein itself, B is the green fluorescence of the secondary antibody after incubation with the positive sample and the secondary antibody, and C is the in situ overlap of red fluorescence and green fluorescence after A and B are overlapped. Thus, it can be shown that the sample is positive.

[0113] Table 1: Comparison of Yo antibody positive paraneoplastic neurologic syndrome antibody detection table

[0114]

[0115] As shown in Table 1, the sensitivity of Example 3 of the present application is obviously better than Comparative Examples 1 to 8, especially at the conventional detection dilution of 1:10 and 1:32, achieving a detection rate of 100%, and compared to Comparative Examples 1 to 3 using CDR2 and / or CDR2L full-length protein, or Comparative Examples 5 to 8 using other truncated design fusion proteins, the detection rate is far lower than that of the present application, and some combinations (Comparative Examples 4, 6 and 7) cannot even be completely detected, thereby proving that the combination of CDR2 (43-158 aa) and CDR2L (159-465 aa) partial sequences selected by the present application is the key to achieving high sensitivity.

[0116] The fixed cells prepared in Example 3 above, Comparative Examples 1 to 8, were added to negative samples (containing 30 healthy human serum samples and 65 serum samples of other antibody-positive paraneoplastic syndrome patients without Yo antibody).

[0117] The serum dilution ratio was 1:10, incubation (incubation at 37°C for 1 hour), PBS washing of the wells 3 times, addition of secondary antibody (sheep anti-human IgG, dilution ratio 1:500) incubation (room temperature for 45 minutes), PBS washing 3 times, addition of a small amount of PBS to soak the cells, and microscopic interpretation.

[0118] The results are shown in Table 2 below: Figure 7 and Table 2 below: Comparison of negative samples.

[0119] Figure 7 The cell immunofluorescence images of the negative samples in Example 3 are shown in the figure, where A is the red fluorescence of the CDR2 and CDR2L fusion protein itself, B is the green fluorescence of the secondary antibody after incubation with the positive sample and the secondary antibody, and C is the overlap of A and B, where the red fluorescence and green fluorescence cannot overlap, thereby indicating that the sample is negative.

[0120] Table 2: Comparison of negative samples

[0121]

[0122] As shown in Table 2, among the 95 negative sample tests, the false positive rate of Example 3 of the present application is 0%, showing excellent specificity. Although Comparative Examples 4 to 8 also achieve the same specificity, their sensitivity is severely insufficient. It should be noted that Comparative Examples 1 to 3 using full-length protein all have different degrees of false positive results, which indicates that by removing the non-specific region of the protein, the present application effectively eliminates cross-reactions while retaining high sensitivity, significantly improving the reliability of the detection results.

[0123] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change of the above embodiment according to the technical essence of the present application falls within the protection scope of the present application.

Claims

1. A recombinant protein for detecting paraneoplastic Yo antibodies by CBA method, characterized in that: The structure of the recombinant protein is in order from N-terminal to C-terminal: a secretion signal peptide, a CDR2 protein partial sequence, a CDR2L protein partial sequence, a connecting peptide A, a transmembrane region, a connecting peptide B and a fluorescent tag, wherein: ​ The secretion signal peptide is a human albumin signal peptide ALB, and the amino acid sequence is shown as SEQ ID NO: 3; The amino acid sequence of the CDR2 protein partial sequence is shown as SEQ ID NO: 1; The amino acid sequence of the CDR2L protein partial sequence is shown as SEQ ID NO: 2; The amino acid sequence of the connecting peptide A is shown as SEQ ID NO: 6; The transmembrane region is a CD8a hinge, and the amino acid sequence is shown as SEQ ID NO: 4; The amino acid sequence of the connecting peptide B is shown as SEQ ID NO: 7; The fluorescent tag is mCherry, and the amino acid sequence is shown as SEQ ID NO:

5.

2. Use of the recombinant protein according to claim 1 for the preparation of a product for the detection of paraneoplastic Yo antibodies, characterized in that: The stable transfected cell obtained by transfecting a host cell with a nucleic acid encoding the recombinant protein for expression is used as a detection product, and the detection of paraneoplastic Yo antibody is performed by CBA method.

3. Use according to claim 2, characterized in that: The host cell includes a CHO cell or a HEK293 cell.

4. A CBA method reagent for detecting paraneoplastic Yo antibodies, characterized in that: The reagent comprises a transfected cell stably expressing the recombinant protein of claim 1.

5. A CBA method kit for detecting paraneoplastic Yo antibodies, characterized in that: The kit comprises the reagent of claim 4.

Images