Recombinant protein and detection reagent for detecting anti-GABABR antibody encephalitis

Abstract

The invention discloses a recombinant protein and a detection reagent for detecting encephalitis of an anti-GABABR antibody, and belongs to the technical field of neuroimmunology diagnos.A GABABR1 partial sequence, a GABABR2 partial sequence and a transmembrane sequence (and a fluorescent label) are adopted to construct a cell strain for stably and highly expressing the recombinant protein by transfecting CHO cells or HEK293 cells, and the recombinant protein and the detection reagent are used for detecting the encephalitis of the anti-GABABR antibody. The sensitivity and the specificity of the kit in anti-GABABR antibody encephalitis detection can be remarkably improved, the aim of detecting the antibody by using a fixed CBA method or a living cell CBA method can be achieved, and the kit is suitable for auxiliary diagnosis of autoimmune encephalitis of cerebrospinal fluid / serum.

Description

Technical Field

[0001] The present invention relates to a recombinant protein and a detection reagent for detecting anti-GABABR antibody encephalitis, and specifically relates to a recombinant protein for detecting anti-GABABR antibody encephalitis by using a cellular immunofluorescence method (CBA method) and a related detection reagent containing the recombinant protein, and belongs to the technical field of neuroimmunology diagnosis. Background Art

[0002] γ-Aminobutyric acid type B receptor (GABAB receptor, GABABR) is an important inhibitory G protein-coupled receptor (GPCR) in the central nervous system. It is composed of two subunits, GABABR1 and GABABR2, which form a functional receptor complex through heterodimerization. Recent studies have found that autoantibodies against GABABR (anti-GABABR antibodies) are closely related to a variety of autoimmune neurological diseases, especially anti-GABABR antibody encephalitis, which has clinical manifestations such as epileptic seizures, cognitive impairment, and mental and behavioral abnormalities. Rapid and accurate detection of anti-GABABR antibodies in patient serum or cerebrospinal fluid is of great significance for the early diagnosis, treatment monitoring, and prognosis evaluation of anti-GABABR antibody encephalitis.

[0003] Traditional methods for detecting anti-GABABR antibody encephalitis include: (1) using recombinant proteins that express a single subunit as antigens, such as single expression of GABABR1 or GABABR2, but their antigenic structure cannot simulate the natural receptor structure, which easily leads to insufficient exposure of antibody binding epitopes, significantly reduced detection sensitivity, and increased false negative rate. (2) Using recombinant proteins that co-express GABABR1 and GABABR2 subunits as antigens to simulate the natural receptor heterodimer structure, but when constructing independent vectors to co-transfect and express GABABR1 and GABABR2, it is difficult to accurately control the expression ratio of the two subunits in the independent expression system, which easily leads to an imbalance in the expression of subunits in the cell population. At the same time, the efficiency of heterodimer formation is affected by transfection conditions (such as plasmid ratio, transfection reagent, cell state, etc.), which significantly increases the uncontrollability of experimental variables. It can be seen that the co-expression of GABABR1 and GABABR2 subunits easily leads to increased batch-to-batch differences in antigenic structure, thereby affecting the stability and repeatability of antibody detection.

[0004] Chinese patent CN102803502A discloses a method for diagnosing and treating autoimmune encephalitis or epilepsy by detecting GABA receptor autoantibodies in biological samples. The patent clearly states that the cause of encephalitis can be determined by detecting autoantibodies against GABAB receptors and their subtypes (including B1 subunits and B2 subunits of GABAB) or GABAB receptor multimers (such as B1 / B2 subunit heterodimers, combinations of B1a / b / c subtypes and B2 subunits). Although the patent proposes a scheme to use GABAB receptor multimers as detection targets, in actual applications, these heteromultimers composed of different subunits still face significant technical challenges during the construction process: one is the uncontrollability of expression: the imbalance of subunit ratios and assembly efficiency are greatly affected by transfection conditions; the second is structural stability: it is difficult to ensure the integrity of the natural conformation of heterodimers; the third is the bottleneck of detection performance: the above factors will directly restrict the uniformity of the antigen structure, thereby affecting the specificity and sensitivity of anti-GABABR antibody encephalitis detection.

[0005] Therefore, how to obtain efficiently expressed and structurally stable GABABR multimeric proteins by optimizing or developing new protein design strategies remains a key research direction for improving detection reliability. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant protein for detecting anti-GABABR antibody encephalitis. By selecting specific truncated sequences of GABABR1 and GABABR2 and obtaining a fusion protein through recombinant expression, the sensitivity and specificity of the fusion protein in detecting anti-GABABR antibody encephalitis can be significantly improved. At the same time, the present invention also provides a detection reagent containing the recombinant antigen, which is suitable for auxiliary diagnosis of autoimmune encephalitis in cerebrospinal fluid / serum.

[0007] The present invention is achieved through the following technical scheme: a recombinant protein for detecting anti-GABABR antibody encephalitis, comprising a partial sequence of GABABR1, a partial sequence of GABABR2 and a transmembrane sequence, The partial sequence of GABABR1 is shown in SEQ ID NO: 1, The partial sequence of GABABR2 is shown in SEQ ID NO: 2. The transmembrane sequence is CD8a hinge.

[0008] It also includes: a first connecting peptide connecting the GABABR1 partial sequence and the GABABR2 partial sequence and a second connecting peptide connecting the GABABR2 partial sequence and the transmembrane sequence.

[0009] The sequence of the first connecting peptide is shown as SEQ ID NO:4, and the sequence of the second connecting peptide is shown as SEQ ID NO:5.

[0010] The transmembrane sequence is connected to a fluorescent label via a third connecting peptide. The fluorescent label is mCherry, The sequence of the third connecting peptide is shown in SEQ ID NO:7.

[0011] A detection reagent for anti-GABABR antibody encephalitis is provided. The nucleic acid encoding the recombinant protein is co-transfected into cells, and the obtained expression cells are used as a detection reagent for anti-GABABR antibody encephalitis.

[0012] The host cells into which the nucleic acid is co-transfected include CHO cells or HEK293 cells.

[0013] The detection format is fixed CBA method or living cell CBA method.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention selectively intercepts partial sequences in the open reading frames of GABABR1 and GABABR2, fuses the selected sequences of the two subunits using a connecting peptide, and introduces a transmembrane domain, so that the antigen epitope can be correctly folded and expressed on the cell membrane surface, thereby achieving the detection sensitivity of the co-transfection of GABABR1 and GABABR2 double subunits, and realizing the purpose of detecting antibodies using a fixed CBA method or a live cell CBA method.

[0015] (2) The present invention has specific truncated sequences of both GABABR1 and GABABR2, which solves the problem of subunit-dependent antibody recognition in which some patient sera only recognize GABABR1 or GABABR2 subunits, while other patients need to rely on the co-expression structure of both, making the detection results more comprehensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the lentiviral vector of the present invention.

[0017] Figure 2 This is the immunofluorescence image of the positive sample in Example 1 of the present invention.

[0018] Figure 3 This is the immunofluorescence image of the negative sample in Example 1 of the present invention.

[0019] Figure 4 This is a comparison diagram of the protein structure and the natural conformation of Example 1 of the present invention. DETAILED DESCRIPTION

[0020] The invention objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0021] 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.

[0022] The present invention uses innovative fusion protein design to intercept partial sequences of GABABR1 and GABABR2 respectively, select connecting sequences to fuse them, and construct a lentiviral expression vector, and then stably transfect CHO cells or HEK293 cells to further screen monoclonal cell lines that stably and highly express recombinant proteins for subsequent detection.

[0023] The technical solution of the present invention can be specifically summarized as follows: 1. Recombinant protein The present invention selectively intercepts a partial sequence of GABABR1 and a partial sequence of GABABR2, and then respectively uses a first connecting peptide linker1, a second connecting peptide linker2 and a transmembrane sequence to recombinantly construct a recombinant protein, which is used as a diagnostic antigen for anti-GABABR antibody encephalitis detection. The recombinant protein design not only retains the key antigenic epitopes of the natural receptor, but also achieves stable expression on the membrane surface through the connecting sequence (i.e., the first connecting peptide linker1, the second connecting peptide linker2 and the transmembrane sequence), and the conformation is complete.

[0024] Specifically, the partial sequence of GABABR1 is derived from the amino acid sequence 1-578 on the N-terminal domain of the transcription isoform GABABR1, and its sequence is shown in SEQ ID NO: 1 as follows: MLLLLLLAPLFLRPPGAGGAQTPNATSEGCQIIHPPWEGGIRYRGLTRDQVKAINFLPVDYEIEYVCRGEREVVGPKVRKCLANGSWTDMDTPSRCVRICSKSYLTLENGKVFLTGGDLPALDGARVDFRCDPDFHLVGSSRSICSQGQWSTPKPHCQVNRTPHSERRAVYIGALFPMSGGWPGGQACQPAVEMALEDVNSRRDILPDYELKLIHHDSKCDPGQATKYLYELLYNDPIKIILMPGCSSVSTLVAEAARMWNLIVLSYGSSSPALSNRQRFPTFFRTHPSATLHNPTRVKLFEKWGWKKIATIQQTTEVFTSLDDLEERVKEAGIEITFRQSFFSDPAVPVKNLKRQDARIIVGLFYETEARKVFCEVYKERLFGKKYVWFLIGWYADNWFKIYDPSINCTVDEMTEAVEGHITTEIVMLNPANTRSISNMTSQEFVEKLTKRLKRHPEETGGFQEAPLAYDAIWALALALNKTSGGGGRSGVRLEDFNYNNQTITDQIYRAMNSSSFEGVSGHVVFDASGSRMAWTLIEQLQGGSYKKIGYYDSTKDDLSWSKTDKWIGGSPPADQTL。

[0025] The partial sequence of GABABR2 is the amino acid sequence from 45 to 469 of GABABR2, and its sequence is as shown in SEQ ID NO: 2 as follows: GAPRPPPSSPPLSIMGLMPLTKEVAKGSIGRGVLPAVELAIEQIRNESLLRPYFLDLRLYDTECDNAKGLKAFYDAIKYGPNHLMVFGGVCPSVTSIIAESLQGWN LVQLSFAATTPVLADKKKYPYFFRTVPSDNAVNPAILKLLKHYQWKRVGTLTQDVQRFSEVRNDLTGVLYGEDIEISDTESFSNDPCTSVKKLKGNDVRIILGQFDQ NMAAKVFCCAYEENMYGSKYQWIIPGWYEPSWWEQVHTEANSSRCLRKNLLAAMEGYIGVDFEPLSSKQIKTISGKTPQQYEREYNNKRSGVGPSKFHGYAYDGIW VIAKTLQRAMETLHASSRHQRIQDFNYTDHTLGRIILNAMNETNFFGVTGQVVFRNGERMGTIKFTQFQDSREVKVGEYNAVADTLEIINDTIRFQGSEPPKDKTI.

[0026] The transmembrane sequence is CD8a hinge, i.e., the amino acid sequence 183-206 of the hinge region of CD8a, and its sequence is shown in SEQ ID NO: 3 as follows: IYIWAPLAGTCGVLLLSLVITLYC.

[0027] The sequence of the first connecting peptide linker1 is shown in SEQ ID NO: 4 as follows: GSTGGGGSGGGGSGGGGSGAASR.

[0028] The sequence of the second connecting peptide linker2 is shown in SEQ ID NO: 5 as follows: ASGGGGSGGGGSSG.

[0029] Furthermore, the present invention forms a traceable fusion expression system by inserting a fluorescent tag into the vector framework of the above-mentioned recombinant protein. For example, the fluorescent tag is connected to the transmembrane sequence of the recombinant vector through the third connecting peptide linker3 to obtain a recombinant protein with a structure of GABABR1 partial sequence-linker1-GABABR2 partial sequence-linker2-CD8a hinge-linker3-mCherry.

[0030] Specifically, the fluorescent tag is mCherry, and its sequence is shown in SEQ ID NO: 6 as follows: MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0031] The sequence of the third connecting peptide linker3 is shown in SEQ ID NO: 7 as follows: AEAAAKEAAAKA.

[0032] 2. Detection reagents The present invention uses the above-mentioned recombinant protein to prepare relevant reagents for detecting anti-GABABR antibody encephalitis, for example, the purified recombinant protein is covalently coupled to fluorescently encoded microbeads to detect anti-GABABR antibody encephalitis in the fixed CBA method; or living cells expressing the recombinant protein are used to detect anti-GABABR antibody encephalitis in the living cell CBA method.

[0033] In a possible embodiment, to construct a lentiviral vector of the recombinant protein GABABR1-GABABR2-CD8a hinge-mCherry, the GABABR1 partial sequence, the GABABR2 partial sequence, the transmembrane sequence CD8a hinge and the fluorescent label mCherry can be sequentially connected through the first connecting peptide linker1, the second connecting peptide linker2 and the third connecting peptide linker3 to form a lentiviral vector. The map of the lentiviral vector is as follows: Figure 1 shown.

[0034] In a possible embodiment, in order to prepare the detection reagent of the above-mentioned recombinant protein, the constructed lentiviral vector can be transfected into CHO cells or HEK293 cells, and monoclonal cells that stably and efficiently express the GABABR1-GABABR2-CD8a hinge-mCherry recombinant protein can be selected, and the cells are mixed with wild-type cells (untransfected CHO or HEK293 cells) in a certain proportion and inoculated into a 96-well plate culture dish. After they grow to a certain confluence, they can be used to prepare the detection reagent used in subsequent immunofluorescence experiments.

[0035] Cell sheet inoculation: CHO-GABABR1-GABABR2-CD8a hinge-mCherry monoclonal cells and control empty cells 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 culture was stopped when the confluence reached 90-100%.

[0036] The grown cell well plates or slides can be directly used for the next immunofluorescence experiment, or the cell slices can be fixed with paraformaldehyde and stored for later use.

[0037] The anti-GABABR antibody encephalitis immunofluorescence detection method of the present invention is as follows: Prepare blood or cerebrospinal fluid samples with sample diluent containing the following: Phosphate buffer (pH 7.0-7.4), BSA (2%); The samples were diluted with sample diluent for the initial determination, the dilution ratio for serum was 1:10, and the original solution (undiluted) was used for cerebrospinal fluid; Sample incubation: 37°C for 1 hour, washed 3 times with PBS; Dilute the secondary antibody with sample diluent: use goat anti-human IgG (H+L) at a dilution ratio of 1:500, incubate at room temperature for 45 minutes, and wash 3 times with PBS; Interpretation: Interpret the positive or negative status of the sample under a microscope.

[0038] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0039] Embodiment 1: In the above manner, the constructed GABABR1 partial sequence (1-578aa) + linker1 + GABABR2 partial sequence (26-469aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry were fused and expressed to detect anti-GABABR antibody encephalitis. The screened stable transfected cells and wild-type CHO cells or HEK293 cells were mixed and plated into 96-well cell culture plates using the fixed CBA method. After 48 hours of culture, they were fixed with paraformaldehyde.

[0040] Comparative Example 1: This comparative example uses the full sequence of GABABR1 (1-961aa) (see NM_001470.4) to express and detect anti-GABABR antibody encephalitis.

[0041] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0042] Comparative Example 2: This comparative example uses the full sequence of GABABR2 (1-941aa) (see NM_005458.8) to express and detect anti-GABABR antibody encephalitis.

[0043] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0044] Comparative Example 3: In this comparative example, the full sequence of GABABR1 (1-961aa) and the full sequence of GABABR2 (1-941aa) were used to construct lentiviral vectors, respectively, and co-expression was used to detect anti-GABABR antibody encephalitis.

[0045] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0046] Comparative Example 4: This comparative example uses the fusion expression of GABABR1 full sequence (1-961aa) + linker1 + GABABR2 full sequence (1-941aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0047] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0048] Comparative Example 5: This comparative example uses the fusion expression of GABABR1 partial sequence (1-578) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0049] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0050] Comparative Example 6: This comparative example uses the fusion expression of GABABR2 partial sequence (26-469aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0051] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0052] Comparative Example 7: This comparative example uses the fusion expression of GABABR1 partial sequence (1-578aa) + linker1 + GABABR2 partial sequence (1-469aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0053] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0054] Comparative Example 8: This comparative example uses the fusion expression of GABABR1 partial sequence (1-578aa) + linker1 + GABABR2 partial sequence (45-469aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0055] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0056] Comparative Example 9: This comparative example uses the fusion expression of GABABR1 partial sequence (1-578aa) + linker1 + GABABR2 partial sequence (26-300aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0057] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0058] Comparative Example 10: This comparative example uses the fusion expression of GABABR1 partial sequence (1-578aa) + linker1 + GABABR2 partial sequence (26-717aa) + linker2 + CD8ahinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0059] A eukaryotic transient expression vector containing the above sequence was constructed and transiently transfected into CHO cells or HEK293 cells using Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde.

[0060] Experimental example: The detection reagents prepared in the above Example 1 and Comparative Examples 1 to 10 were added to positive samples clinically diagnosed as anti-GABABR antibody encephalitis, and 328 samples confirmed to be anti-GABABR antibody encephalitis were selected according to the diagnostic criteria of GABABR encephalitis expert consensus.

[0061] The inclusion of samples must meet three conditions: A, B and C: A. One or more of the following six main symptoms: (1) refractory epilepsy; (2) cognitive dysfunction; (3) mental and behavioral abnormalities; (4) impaired consciousness; (5) movement disorders; (6) autonomic dysfunction; B. Anti-GABABR antibody encephalitis positive: It is recommended to use the cerebrospinal fluid CBA antibody positive. If only serum samples are available for testing, in addition to the positive CBA result, TBA is also required for final confirmation, and low titer serum positivity (1:10) is not diagnostic.

[0062] C. Reasonably exclude other causes.

[0063] The samples were serum and cerebrospinal fluid, where the dilution ratios of serum were 1:10, 1:32, and 1:100; the cerebrospinal fluid was the original solution without dilution.

[0064] Immunofluorescence was used for detection, and the results are shown in Figure 2 and Table 1 below: Comparison of detection rates of GABABR antibody disease positive samples.

[0065] Figure 2 This is the immunofluorescence image of the positive sample in Example 1, in which A is the red light of the GABABR1 and GABABR2 fusion protein itself, B is the green fluorescence of the secondary antibody after the positive sample and the secondary antibody are incubated, and C is the overlap of the red and green lights in situ, which indicates that the sample is detected as positive.

[0066] Table 1: Comparison of detection rates of GABABR antibody disease positive samples Depend on Figure 2 As shown in Table 1, Example 1 using the method of the present invention can well detect anti-GABABR antibody encephalitis positive samples, and when compared with the test results of Comparative Examples 1 to 10, it has better sensitivity in the case of a higher serum dilution multiple. Comparative Example 2 does not contain an antigen epitope and cannot be detected, and Comparative Examples 6-10 cannot be detected because the selected sequence fragments of the fusion protein change the spatial conformation of the protein.

[0067] The fixed cells prepared in the above Example 1 and Comparative Examples 1 to 10 were added to 24 serum samples of healthy subjects, 45 serum samples of other patients with non-anti-GABABR antibody encephalitis, and 38 cerebrospinal fluid samples, respectively.

[0068] Immunofluorescence was used for detection, and the results are shown in Figure 3 And the following table 2: Comparison of negative sample detection rates in healthy people.

[0069] Figure 3 This is the immunofluorescence image of the negative sample in Example 1, in which A is the red light of the GABABR1 and GABABR2 fusion protein itself, B is the negative sample without green fluorescence after incubation with the secondary antibody, and C is the superposition of A and B, which shows that the sample detection is negative.

[0070] Table 2: Comparison of negative sample detection rates in healthy individuals Further, by Figure 3 As can be seen from Table 2, Example 1 using the method of the present invention can detect negative samples very well, and the specificity is in compliance.

[0071] In summary, the method of the present invention can be used for the detection of GABABR autoimmune antibodies by adopting the fusion expression of specific partial sequences in GABABR1 and GABABR2, has specificity and sensitivity, and its detection effect is better than the methods of Comparative Examples 1 to 10. In Comparative Examples 1 to 10, Comparative Examples 1 and 2 are detection methods for expressing the full length of GABABR (GABABR1 or GABABR2 transcript) alone, Comparative Examples 3 and 4 are methods for fusion expression of the full length of GABABR1 and GABABR2, Comparative Examples 5 and 6 are methods for expressing the partial sequences of GABABR1 and GABABR2 in Example 1, respectively, and Comparative Examples 7 to 10 are methods for fusion expression of partial sequences of GABABR1 and GABABR2, but their fusion sequences are not the same, wherein GABABR1 is the same as Example 1, and the partial sequence of GABABR2 is longer or shorter than that of Example 1.

[0072] It should be further noted that the present invention utilizes specific truncated GABABR1 and GABABR2 sequences to construct recombinant proteins, which can enable the antigen epitopes to fold correctly and present a natural conformation (see Figure 4 , Figure 4 Wherein A is the protein structure of Example 1, B is the native conformation, and C is the comparison after A and B are overlapped). Therefore, when used for anti-GABABR antibody encephalitis detection, it has significant specificity and sensitivity. However, since Comparative Examples 1 to 10 have different selected sequences, their respective conformational folding and expression levels are also different, and they cannot provide a detection effect equivalent to that of the present invention. Comparative Examples 1 and 2 respectively selected the full sequences of GABABR1 and GABABR2, but their extracellular domains were incomplete compared to the natural conformation; Comparative Example 3 co-expressed the full sequences of GABABR1 and GABABR2, but the co-transfection ratio was difficult to control and the expression level was poor; Comparative Example 4 constructed the full sequences of GABABR1 and GABABR2 into an expression vector, and the sequence was too long and the expression level was low; Comparative Example 5 used the extracellular domain of GABABR1, and Comparative Example 6 used the extracellular domain of GABABR2, and their conformations were incomplete compared to the natural conformation; Comparative Examples 7 to 10 used the extracellular domain of GABABR1 and different partial sequences of GABABR2, and their structures were different from the natural conformation.

[0073] 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 for detecting anti-GABABR antibody encephalitis, characterized in that: Contains a partial sequence of GABABR1, a partial sequence of GABABR2 and a transmembrane sequence, The partial sequence of GABABR1 is shown in SEQ ID NO: 1, The partial sequence of GABABR2 is shown in SEQ ID NO:

2. The transmembrane sequence is CD8a hinge.

2. The recombinant protein according to claim 1, characterized in that: Also includes: A first connecting peptide connecting the GABABR1 partial sequence and the GABABR2 partial sequence and a second connecting peptide connecting the GABABR2 partial sequence and the transmembrane sequence.

3. The recombinant protein according to claim 2, characterized in that: The sequence of the first connecting peptide is shown in SEQ ID NO:4, and the sequence of the second connecting peptide is shown in SEQ ID NO:

5.

4. The recombinant protein according to claim 1, characterized in that: The transmembrane sequence is connected to a fluorescent label via a third connecting peptide. The fluorescent label is mCherry, The sequence of the third connecting peptide is shown in SEQ ID NO:

7.

5. A detection reagent for anti-GABABR antibody encephalitis, characterized in that: The nucleic acid encoding the recombinant protein according to any one of claims 1 to 4 is co-transfected into cells, and the obtained expression cells are used as a detection reagent for anti-GABABR antibody encephalitis.

6. The detection reagent according to claim 5, characterized in that: The host cells into which the nucleic acid is co-transfected include CHO cells or HEK293 cells.

7. The detection reagent according to claim 5, characterized in that: The detection format is fixed CBA method or living cell CBA method.

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