A recombinant protein and its application in anti-Hu antibody using cell immunofluorescence method

Abstract

The present invention discloses a recombinant protein and its application in anti-Hu antibody by cell immunofluorescence method, belonging to the field of biomedical engineering technology, wherein the recombinant protein is a fusion protein obtained by fusing a partial sequence of HuD protein, a partial sequence of HuC protein and a partial sequence of HuB protein with a secretion signal peptide, a linker, a transmembrane sequence and a fluorescent tag, and its sequence is shown in SEQ ID NO: 1. The recombinant protein provided by the present invention is obtained by redesigning and fusion expressing partial sequences of HuD, HuC and HuB proteins, and the fusion-expressed protein can be stably overexpressed on the cell membrane of eukaryotic cells, thereby achieving high accuracy and high sensitivity of anti-Hu antibody detection in cell immunofluorescence method (CBA method).

Description

Technical Field

[0001] The present invention relates to a recombinant protein and its application in anti-Hu antibody by cell immunofluorescence method, specifically to a recombinant protein which fuses partial sequences of HuD, HuC and HuB proteins and can be expressed on the cell membrane of eukaryotic cells, and the application of the recombinant protein in anti-Hu antibody detection by cell immunofluorescence method (CBA method), belonging to the field of biomedical engineering technology. Background Art

[0002] Paraneoplastic neurological syndrome (PNS) is a rare neurological disease associated with malignant tumors. It is generally believed that antibodies against ectopic antigens expressed by tumor cells cross-react with antigens of the nervous system and trigger an immune cascade reaction, leading to neurodegeneration. So far, several "well-characterized" anti-neuronal antibodies have been identified (Hu, Ri, Yo, CV2 / CRMP5, Ma1, Ma2 / Ta, and Amphiphysin, etc.). In the known published diagnostic criteria for PNSs, PNSs is defined as: ① It can affect any part of the nervous system, often showing stereotyped clinical manifestations; ② It is associated with malignant tumors; ③ It is a neurological disease with immune-mediated pathogenesis. This definition clearly pointed out for the first time the causal relationship between malignant tumors and PNSs in PNSs and the role of immune mechanisms in its pathogenesis, and provided a basis for the early diagnosis of PNSs patients and the formulation of scientific and reasonable tumor screening and antibody detection programs for suspected PNSs patients. Among them, the correlation between antibodies and PNSs shows that antibodies are crucial for guiding the search for potential tumors. Three groups of antibodies were defined based on the frequency of their association with malignant tumors: the first group: "high-risk antibodies, defined as antibodies that frequently appear (>70%) in potential cancer patients; the second group: "medium-risk antibodies, defined as antibodies that are associated with malignant tumors in 30-70% of cases; the third group: "low-risk antibodies, antibodies with a much lower correlation with malignant tumors, among which anti-Hu antibodies are high-risk antibodies.

[0003] Anti-Hu antibodies were first discovered in patients with small cell lung cancer (SCLC) and subacute sensory neuropathy in 1985 and have been shown to be the most common paraneoplastic antibodies. Currently, anti-Hu antibodies are mainly detected by rodent brain tissue immunohistochemistry or immunofluorescence, plus recombinant protein immunoblotting. However, in the clinical testing of anti-Hu antibodies, due to many laboratory conditions, immunohistochemistry cannot be used for simultaneous detection. At the same time, it should be pointed out that the use of rodent brain tissue immunohistochemistry cannot achieve a high detection accuracy due to its heterogeneity; and only using protein blotting for detection, according to the literature comparison of the positive results of single immunoblotting and the consistency of the diagnosis of anti-Hu paraneoplastic syndrome, different commercial brands of immunoblotting can only achieve a diagnosis rate of 65-88%.

[0004] At present, the immunoblotting method for detecting anti-Hu antibodies usually uses recombinant human HuD (gene: ELAVL4) protein as an antigen, see Chinese patents CN113252906A and CN114441769A; other literature also discloses that HuC (gene: ELAVL3) and HuB (gene: ELAVL2) can also be used for anti-Hu antibody detection, and the median titer of HuC is higher than that of HuD, see "Hu Antigens: Reactivity with Hu Antibodies, Tumor Expression, and Major Immunogenic Sites", Neurol 1995; "Hu Antigen Specificities of ANNA-I Autoantibodies in Paraneoplastic Neurological Disease", Journal of autoimmunity 1999; and "Anti-HuC and -HuD "Autoantibodies are differential sero-diagnostic markers for small cell carcinoma from large cell neuroendocrine carcinoma of the lung", International journal of oncology, 2012 (Anti-HuC and -HuD autoantibodies are differential sero-diagnostic markers for small cell carcinoma from large cell neuroendocrine carcinoma of the lung, International journal of oncology, 2012).

[0005] However, since HuD, HuC, and HuB are all neural-specific RNA-binding proteins, their expression sites are located in the cell nucleus, and the full-length nuclear protein gene is directly used for eukaryotic cell nucleus localization expression, there is not only a risk of gene silencing, but also increases the difficulty of direct and stable expression of nuclear proteins. Therefore, for the anti-Hu antibody with the highest detection rate in the series of antibodies for paraneoplastic nervous system syndromes, it is of great significance to provide a recombinant protein technology that can detect anti-Hu antibodies with high efficiency and stable expression in cells and improve its detection accuracy and sensitivity. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant protein and its application in anti-Hu antibody by cellular immunofluorescence method. The recombinant protein is obtained by redesigning and fusion expressing partial sequences of HuD, HuC and HuB proteins. The fusion-expressed protein can be stably overexpressed on the cell membrane of eukaryotic cells, thereby achieving high accuracy and high sensitivity in anti-Hu antibody detection in cellular immunofluorescence method (CBA method).

[0007] The present invention is achieved through the following technical solutions:

[0008] (I) The present invention provides a recombinant protein, which is a fusion protein obtained by fusing a partial sequence of HuD protein, a partial sequence of HuC protein and a partial sequence of HuB protein with a secretion signal peptide, a linker, a transmembrane sequence and a fluorescent tag, and its sequence is shown in SEQ ID NO: 1.

[0009] The partial sequence of the HuD protein is shown in SEQ ID NO: 2; the partial sequence of the HuC protein is shown in SEQ ID NO: 3; and the partial sequence of the HuB protein is shown in SEQ ID NO: 4.

[0010] The secretory signal peptide is human albumin signal peptide ALB, and its sequence is shown in SEQ ID NO:5.

[0011] The transmembrane sequence is a partial sequence of hCNTNAP2, and its sequence is shown in SEQ ID NO:6.

[0012] The linker includes a linker peptide 1 connecting a partial sequence of the HuD protein and a partial sequence of the HuC protein, a linker peptide 2 connecting a partial sequence of the HuB protein and a transmembrane sequence, and a linker peptide 3 connecting the transmembrane sequence and a fluorescent label.

[0013] The sequence of the connecting peptide 1 is shown in SEQ ID NO: 7; the sequence of the connecting peptide 2 is shown in SEQ ID NO: 8; and the sequence of the connecting peptide 3 is shown in SEQ ID NO: 9.

[0014] The fluorescent tag is mCherry, and its sequence is shown in SEQ ID NO:10.

[0015] (ii) The present invention provides the use of the above-mentioned recombinant protein in anti-Hu antibodies using the cell-based immunofluorescence assay, namely: using the cell-based immunofluorescence assay (CBA method), the nucleic acid encoding the recombinant protein is transfected into host cells for expression, and the co-transfected cells obtained are used as detection products for the detection of anti-Hu antibodies.

[0016] The host cells include CHO cells or 293 cells (HEK293 cells).

[0017] The detection product includes a detection reagent or a detection kit.

[0018] Furthermore, the present invention can also provide an anti-Hu antibody detection product, which is suitable for detecting anti-Hu antibodies by cellular immunofluorescence assay (CBA method), and the detection product is prepared by co-transfected cells obtained by transfecting host cells with nucleic acid encoding the above-mentioned recombinant protein for expression.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] (1) The present invention selects partial sequences of HuD, HuC and HuB proteins, and fuses them by adding signal peptides, linker peptides, transmembrane sequences and fluorescent tags, so that the antigenic determining regions of HuD, HuC and HuB proteins are fused into recombinant proteins, which can be localized on the cell membrane and achieve stable overexpression outside the cell membrane. Localization on the cell membrane avoids gene silencing that may be caused by overexpression of nuclear proteins in the cell nucleus, and also allows the use of fixed non-permeabilized immunofluorescence method or live cell method to detect samples, thereby improving the detection sensitivity.

[0021] (2) The present invention uses fusion expression of HuD, HuC and HuB, that is, extracting partial sequences of specific regions on HuD, HuC and HuB for fusion expression. This method is significantly superior to the CBA method that uses the full-length expression of HuD, HuC or HuB alone in terms of the number and specificity of antigenic epitopes. Experiments have shown that the fusion protein of the present invention is more sensitive to the detection titer of positive samples.

[0022] (3) The present invention is suitable for detecting anti-Hu antibodies by the CBA method. Compared with the immunoblotting method, the antigen protein in the CBA method is not denatured, and the natural conformation of the protein is maintained, which can more accurately detect positive samples and reduce false positive results.

[0023] (4) In the prior art, when a recombinant protein is expressed in the nucleus, the cells need to be permeabilized before detection so that the antibody can enter the cells and bind to them. However, the present invention does not require the cells to be permeabilized. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a reference schematic diagram of the recombinant protein of the present invention being localized on the cell membrane.

[0025] Figure 2 The figure is a map of the lentiviral vector of the present invention.

[0026] Figure 3 This is an immunofluorescence image of the anti-Hu antibody detection of the present invention (positive sample).

[0027] Figure 4 This is an immunofluorescence image of the anti-Hu antibody detection of the present invention (negative sample). DETAILED DESCRIPTION

[0028] The invention objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0029] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0030] The present invention aims to provide a fusion protein of HuD, HuC and HuB that can be stably overexpressed on the cell membrane of eukaryotic cells. The fusion protein is obtained by fusing a partial sequence of the HuD protein, a partial sequence of the HuC protein and a partial sequence of the HuB protein with a secretion signal peptide, a linker, a transmembrane sequence and a fluorescent label. The fusion protein can replace the existing method of expressing the full-length HuD, HuC or HuB in the cell nucleus alone, which not only overcomes the risk of gene silencing, but also improves the accuracy and sensitivity of anti-Hu antibody detection, and is suitable for CBA method to detect anti-Hu antibodies, which can more accurately detect positive samples and reduce false positive results.

[0031] In order to achieve the above technical effects, the key points of the technical solution of the present invention are:

[0032] 1) Construct new recombinant proteins that can be efficiently and stably expressed in cells;

[0033] 2) The novel recombinant protein can specifically bind to anti-Hu antibodies and can be used to detect the presence of anti-Hu antibodies in biological samples;

[0034] 3) There is no need to permeabilize the cells before testing.

[0035] Further, the technical solution of the present invention can be summarized as follows:

[0036] The fusion protein is the recombinant protein of the present invention, and its sequence is shown in SEQ ID NO: 1, as follows:

[0037] .

[0038] The fusion protein is a recombinant protein that co-expresses ALB-ELAVL4 partial sequence-connector peptide 1-ELAVL3 partial sequence-ELAVL2 partial sequence-connector peptide 2-hCNTNAP2 partial sequence-connector peptide 3-mCherry, which can achieve stable overexpression on the cell membrane of eukaryotic cells, see Figure 1 shown.

[0039] The partial sequence of HuD protein, namely the partial sequence of ELAVL4, is from the amino acid sequence 45-211 on the N-terminal domain of the ELAVL4 transcript isoform NM_021952.5, and its sequence is shown in SEQ ID NO: 2, as follows:

[0040] KTNLIVNYLPQNMTQEEFRSLFGSIGEIESCKLVRDKITGQSLGYGFVNYIDPKDAEKAINTLNGLRLQTKTIKVSYARPSSASIRDANLYVSGLPKTMTQKELEQLFSQYGRIITSRILVDQVTGVSRGVGFIRFDKRIEAEEAIKGLNGQKPSGATEPITVKFAN.

[0041] The partial sequence of HuC protein, i.e. the partial sequence of ELAVL3, is derived from the amino acid sequence 39-123 on the N-terminal domain of the ELAVL3 transcript isoform NM_001420.4, and its sequence is shown in SEQ ID NO: 3, as follows:

[0042] NLIVNYLPQNMTQDEFKSLFGSIGDIESCKLVRDKITGQSLGYGFVNYSDPNDADKAINTLNGLKLQTKTIKVSYARPSSASIR.

[0043] The partial sequence of HuB protein, i.e. the partial sequence of ELAVL2, is derived from the amino acid sequence 124-205 on the N-terminal domain of the ELAVL2 transcript isoform NM_004432.5, and its sequence is shown in SEQ ID NO: 4, as follows:

[0044] DANLYVSGLPKTMTQKELEQLFSQYGRIITSRILVDQVTGISRGVGFIRFDKRIEAEEAIKGLNGQKPPGATEPITVKFANN.

[0045] The secretory signal peptide is human albumin signal peptide ALB, and its sequence is shown in SEQ ID NO: 5, as follows:

[0046] MKWVTFISLLFLFSSAYS.

[0047] The transmembrane sequence is a partial sequence of hCNTNAP2, which is derived from the amino acid sequence 1263-1331 of the hCNTNAP2 transcript isoform NM_014141.6, and its sequence is shown in SEQ ID NO: 6 as follows:

[0048] IIGGVIAVVIFTILCTLVFLIRYMFRHKGTYHTNEAKGAESAESADAAIMNNDPNFTETIDESKKEWLI.

[0049] The linker includes a linker peptide 1 connecting the ELAVL3 partial sequence and the ELAVL2 partial sequence, a linker peptide 2 connecting the ELAVL2 partial sequence and the hCNTNAP2 partial sequence, and a linker peptide 3 connecting the hCNTNAP2 partial sequence and the fluorescent tag.

[0050] Specifically, the sequence of connecting peptide 1 is shown in SEQ ID NO: 7, which is AEAAAKEAAAKA; the sequence of connecting peptide 2 is shown in SEQ ID NO: 8, which is PAPAP; and the sequence of connecting peptide 3 is shown in SEQ ID NO: 9, which is SGGGGSGGGGSGGGGS.

[0051] The fluorescent tag is mCherry, and its sequence is shown in SEQ ID NO: 10, as follows:

[0052] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0053] The present invention can also use the above-mentioned recombinant protein for the detection of anti-Hu antibodies in a cell immunofluorescence method, that is, the co-transfected cells obtained by transfecting CHO cells or HEK293 cells with the nucleic acid encoding the recombinant protein for expression are used as detection products to detect anti-Hu antibodies. The cell immunofluorescence method can be a fixed CBA method or a live cell CBA method.

[0054] At the same time, the present invention also provides a detection product for detecting anti-Hu antibodies used in the fixed CBA method or the living cell CBA method. The detection product can be a detection reagent or a detection kit, which is specifically prepared by co-transfected cells obtained by transfecting CHO cells or HEK293 cells with nucleic acid encoding the recombinant protein for expression.

[0055] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0056] Example 1: Construction of a lentiviral vector for recombinant protein

[0057] The ALB signal peptide, the partial sequence of ELAVL4, the partial sequence of ELAVL3, the partial sequence of ELAVL2, the hCNTNAP2 transmembrane sequence and the fluorescent label mCherry are sequentially connected through three connecting peptides to form a lentiviral vector, namely:

[0058] ALB-ELAVL4 partial sequence-connector peptide 1-ELAVL3 partial sequence-ELAVL2 partial sequence-connector peptide 2-hCNTNAP2 partial sequence-connector peptide 3-mCherry.

[0059] The sequences thereof are shown in SEQ ID NO: 1 to SEQ ID NO: 10, and the map of the lentiviral vector constructed therefrom is shown in Figure 2 shown.

[0060] Example 2: Preparation of anti-Hu antibody detection reagent

[0061] The lentiviral vector prepared in Example 1 was transfected into CHO or 293 cells, and the transfected cells were screened by limiting dilution to obtain monoclonal cells expressing ELAVL4-ELAVL3-ELAVL2-hCNTNAP2-mCherry fusion protein, and then the culture dish full of transfected ELAVL4-ELAVL3-ELAVL2-hCNTNAP2-mCherry cells was taken, digested with trypsin (trypsin-EDTA) solution to form a single cell suspension, and then the cell density was calculated with a cell counting plate, and then the cells were diluted with complete culture medium, and the cells were diluted to 4 / 100μL, 2 / 100μL, and 1 / 100μL by limiting dilution. Then, 100μL / well were inoculated into 96-well plates, and the wells of single cell groups were screened out, and the wells with strong red fluorescence were selected for freezing and primary antibody immunofluorescence detection. The cells that meet the requirements can be used to prepare detection reagents for subsequent immunofluorescence experiments.

[0062] Cell sheet inoculation: CHO-ELAVL4-ELAVL3-ELAVL2-hCNTNAP2-mCherry monoclonal cells and control empty cells after devitalization were mixed evenly at a ratio of monoclonal cells to control cells = 1:2, and the total density was 1×2×10 5 / mL was inoculated into 96-well plates or cell slides, cultured with F12K+10% FBS+NEAA, grown for about 48 hours, and the culture was stopped when the confluence reached 90-100%.

[0063] The living cell method can be used to directly use the grown cell well plate or slide for the next immunofluorescence experiment, or the cell sheet can be fixed with paraformaldehyde and stored for later use.

[0064] Embodiment 3:

[0065] According to the methods of Examples 1 and 2, the constructed ALB-ELAVL4 (45-211aa)-connector peptide 1-ELAVL3 (39-123aa)-ELAVL2 (124-205aa)-connector peptide 2-hCNTNAP2 (1263-1331aa)-connector peptide 3-mCherry was fused and expressed to detect anti-Hu antibodies. The screened stable transfected cells and wild-type CHO or HEK293 cells were mixed and plated into a 96-well cell culture plate using the fixed CBA method. After culturing for 48 hours, the cells were fixed with paraformaldehyde.

[0066] The fixed CBA method of this embodiment is to immerse CHO or HEK293 cells in a fixative containing paraformaldehyde (the concentration ratio of paraformaldehyde, NaCl, KCl, Na2HPO4, KH2PO4 and glycyl-L-glutamate in the fixative is 10:60:1.5:10.8:1.8:1, and the pH value of the fixative is 7.0-7.6) and fix them on a cell culture plate for 20 minutes, then wash the cell sheet with phosphate buffer for 3 times, each time for 5 minutes, and after washing, add blocking solution (phosphate buffer and 5% bovine serum albumin) to the wells of the cell culture plate, and block at room temperature for 1 hour.

[0067] Comparative Example 1:

[0068] In this comparative example, the full sequence of ELAVL4 (see NM_021952.5) ​​was used to express and detect anti-Hu antibodies, and the paraformaldehyde-fixed cell sheet was obtained by the fixed CBA method (same as Example 3).

[0069] Specifically referring to Example 1 and Example 2, the full sequence of ELAVL4 and the fluorescent tag mCherry were connected via a connecting peptide to form a lentiviral vector, and the full sequence of ELAVL4-connecting peptide 1-mCherry was constructed to express and detect anti-Hu antibodies.

[0070] The full sequence of ELAVL4 is shown in SEQ ID NO: 11, as follows:

[0071] MVMIISTMEPQVSNGPTSNTSNGPSSNNRNCPSPMQTGATTDDSKTNLIVNYLPQNMTQEEFRSLFGSIGEIESCKLVRDKITGQSLGYGFVNYIDPKDAEKAINTLNGLRLQTKTIKVSYARPSSASIRDANLYVSGLPKTMTQKELEQLFSQYGRIITSRILVDQVTGVSRGVGFIRFDKRIEAEEAI KGLNGQKPSGATEPITVKFANNPSQKSSQALLSQLYQSPNRRYPGPLHHQAQRFRLDNLLNMAYGVKRLMSGPVPPSACPPRFSPITIDGMTSLVGMNIPGHTGTGWCIFVYNLSPDSDESVLWQLFGPFGAVNNVKVIRDFNTNKCKGFGFVTMTNYDEAAMAIASLNGYRLGDRVLQVSFKTNKAHKS.

[0072] The connecting peptide 1 is shown as SEQ ID NO:7, and mCherry is shown as SEQ ID NO:10.

[0073] Comparative Example 2:

[0074] In this comparative example, the full sequence of ELAVL3 (see NM_001420.4) was used to express and detect anti-Hu antibodies, and the paraformaldehyde-fixed cell sheet was obtained by the fixed CBA method (same as Example 3).

[0075] Specifically referring to Example 1 and Example 2, the full sequence of ELAVL3 and the fluorescent tag mCherry were connected via a connecting peptide to form a lentiviral vector, and the full sequence of ELAVL3-connecting peptide 1-mCherry was constructed to express and detect anti-Hu antibodies.

[0076] The full sequence of ELAVL3 is shown in SEQ ID NO: 12, as follows:

[0077] MVTQILGAMESQVGGGPAGPALPNGPLLGTNGATDDSKTNLIVNYLPQNMTQDEFKSLFGSIGDIESCKLVRDKITGQSLGYGFVNYSDPNDADKAINTLNGLKLQTKTIKVSYARPSSASIRDANLYVSGLPKTMSQKEMEQLFSQYGRIITSRILVDQVTGVSRGVGFIRFDKRIEAEEAIK GLNGQKPLGAAEPITVKFANNPSQKTGQALLTHLYQSSARRYAGPLHHQTQRFRLDNLLNMAYGVKSPLSLIARFSPIAIDGMSGLAGVGLSGGAAGAGWCIFVYNLSPEADESVLWQLFGPFGAVTNVKVIRDFTTNKCKGFGFVTMTNYDEAAMAIASLNGYRLGERVLQVSFKTSKQHKA.

[0078] The connecting peptide 1 is shown as SEQ ID NO:7, and mCherry is shown as SEQ ID NO:10.

[0079] Comparative Example 3:

[0080] In this comparative example, the full sequence of ELAVL2 (see NM_004432.5) was used to express and detect anti-Hu antibodies, and the paraformaldehyde-fixed cell sheet was obtained by the fixed CBA method (same as Example 3).

[0081] Specifically referring to Example 1 and Example 2, the full sequence of ELAVL2 and the fluorescent tag mCherry were connected via a connecting peptide to form a lentiviral vector, and the full sequence of ELAVL2-connecting peptide 1-mCherry was constructed to express and detect anti-Hu antibodies.

[0082] The full sequence of ELAVL2 is shown in SEQ ID NO: 13, as follows:

[0083] METQLSNGPTCNNTANGPTTINNNCSSPVDSGNTEDSKTNLIVNYLPQNMTQEELKSLFGSIGEIESCKLVRDKITGQSLGYGFVNYIDPKDAEKAINTLNGLRLQTKTIKVSYARPSSASIRDANLYVSGLPKTMTQKELEQLFSQYGRIITSRILVDQVTGISRGVGFIRFDKRIEAE EAIKGLNGQKPPGATEPITVKFANNPSQKTNQAILSQLYQSPNRRYPGPLAQQAQRFRLDNLLNMAYGVKRFSPMTIDGMTSLAGINIPGHPGTGWCIFVYNLAPDADESILWQMFGPFGAVTNVKVIRDFNTNKCKGFGFVTMTNYDEAAMAIASLNGYRLGDRVLQVSFKTNKTHKA.

[0084] The connecting peptide 1 is shown as SEQ ID NO:7, and mCherry is shown as SEQ ID NO:10.

[0085] Comparative Example 4:

[0086] In this comparative example, the full sequences of ELAVL4, ELAVL3 and ELAVL2 were used to construct ALB-ELAVL4-connector peptide 1-ELAVL3-ELAVL2-connector peptide 2-hCNTNAP2 (1263-1331aa)-connector peptide 3-mCherry to express and detect anti-Hu antibodies, and the fixed CBA method (same as Example 3) was used to obtain polyformaldehyde fixed cell sheets.

[0087] Comparative Example 5:

[0088] In this comparative example, the full sequences of ELAVL4 (45-245aa) and ELAVL3 and ELAVL2 were used to construct ALB-ELAVL4 (45-245aa)-connector peptide 1-ELAVL3-ELAVL2-connector peptide 2-hCNTNAP2 (1263-1331aa)-connector peptide 3-mCherry to express and detect anti-Hu antibodies, and the paraformaldehyde-fixed cell sheet was obtained by the CBA fixation method.

[0089] ELAVL4 (45-245aa) is derived from the amino acid sequence 45-245 on the N-terminal domain of the ELAVL4 transcript isoform NM_021952.5, and its sequence is shown in SEQ ID NO: 14, as follows:

[0090] KTNLIVNYLPQNMTQEEFRSLFGSIGEIESCKLVRDKITGQSLGYGFVNYIDPKDAEKAINTLNGLRLQTKTIKVSYARPSSASIRDANLYVSGLPKTMTQKELEQLFSQYGRIITSRILVDQVTGVSRGVGFIRFDKRIEAEEAIKGLNGQKPSGATEPITVKFANNPSQKSSQALLSQLYQSPNRRYPGPLHHQAQRFR.

[0091] The fixed cell sheets prepared in Example 3 and Comparative Examples 1 to 5 were subjected to the following tests:

[0092] (1) PNS positive sample detection

[0093] Inclusion criteria for PNS-positive samples: clinical patients with a PNS-Care Score ≥ 8, for details, please refer to "NeurolNeuroimmunol Neuroinflamm", "Updated Diagnostic Criteria for ParaneoplasticNeurologic Syndromes", Volume 8, Issue 4 (NeurolNeuroimmunol Neuroinflammation, "New Diagnostic Criteria for Paraneoplastic Neurological Syndromes", Volume 8, Issue 4).

[0094] Sources of PNS-positive samples: clinical test specimens (human serum and cerebrospinal fluid samples), with a total of 45 samples, including 12 cerebrospinal fluid samples and 33 serum samples.

[0095] Detection method: Prepare sample diluent, the specific formula is as follows: add 1% bovine serum albumin to phosphate buffer, filter and sterilize after dissolving, dilute human serum and cerebrospinal fluid samples with sample diluent, use original solution (undiluted) for cerebrospinal fluid, and dilute serum samples at a ratio of 1:10. Add the diluted samples to the fixed cell culture well plate, incubate at 37°C for 1 hour, discard the sample solution, and wash the cell sheet with phosphate buffer 3 times, 5 minutes each time. Then, dilute the secondary antibody with the same sample diluent, the secondary antibody is Delight 488-labeled goat anti-human IgG (Fc), the dilution ratio is 1:500, incubate at room temperature for 40 minutes, discard the secondary antibody, wash the cell sheet with phosphate buffer 3 times, 5 minutes each time, add a little phosphate buffer to completely immerse the cell sheet, and observe under a fluorescence microscope.

[0096] The test results are shown in Table 1. As can be seen from the results in Table 1, the positive detection rate of Example 3 can reach 100%, which is significantly better than the positive detection rates of Comparative Examples 1 to 5.

[0097] Table 1

[0098]

[0099] (2) Specificity detection

[0100] A total of 58 clinical test specimens were collected, including 30 healthy control sera and 28 sera from patients with other PNS antibody-positive and anti-Hu antibody-negative results, and the specimens were tested according to the above-mentioned PNS-positive sample detection method.

[0101] The test results are shown in Table 2. As can be seen from the results in Table 2, the specificity of Example 3 is significantly better than that of Comparative Examples 1 to 5.

[0102] Table 2

[0103]

[0104] Figure 3 This is an immunofluorescence diagram of anti-Hu antibody (positive sample) detection using Example 3. Figure 3 A in the figure represents the red fluorescence of the recombinant protein itself, B represents the green fluorescence of the secondary antibody, and C represents the overlap of red and green light. Figure 4 This is an immunofluorescence diagram of anti-Hu antibody (negative sample) detection using Example 3. Figure 4 A represents the red fluorescence of the recombinant protein itself, B represents the green fluorescence of the secondary antibody, and C represents the overlap of red and green light. Figure 3 and Figure 4 It can be seen that the recombinant protein of Example 3 has a high fluorescence contrast and no specific fluorescence background during immunofluorescence detection.

[0105] (3) Sensitivity test

[0106] 23 clinical test specimens were collected, all of which were anti-Hu antibody PNS-positive serum samples. The samples were tested according to the above-mentioned PNS-positive sample detection method, and the serum dilution ratios were 1:10, 1:32, and 1:100, respectively.

[0107] The test results are shown in Table 3. As can be seen from the results in Table 3, the detection sensitivity of Example 3 is significantly better than that of Comparative Examples 1 to 5.

[0108] Table 3

[0109]

[0110] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that: The recombinant protein is a fusion protein obtained by fusing a partial sequence of HuD protein, a partial sequence of HuC protein and a partial sequence of HuB protein with a secretion signal peptide, a linker, a transmembrane sequence and a fluorescent tag. The sequence of the recombinant protein is shown in SEQ ID NO:

1.

2. The use of the recombinant protein as claimed in claim 1 in the preparation of a detection product for detecting anti-Hu antibodies by cellular immunofluorescence method, characterized in that: The cell immunofluorescence method is used to detect anti-Hu antibodies by transfecting the nucleic acid encoding the recombinant protein into host cells for expression and obtaining co-transfected cells as detection products.

3. The use according to claim 2, characterized in that: The host cells include CHO cells or 293 cells.

4. The use according to claim 2, characterized in that: The detection product includes a detection reagent or a detection kit.

Images