A recombinant protein and a detection reagent for detecting anti-GABABR antibody encephalitis

Abstract

The present invention discloses a recombinant protein and a detection reagent for detecting anti-GABABR antibody encephalitis, belonging to the technical field of neuroimmunological diagnosis. By using partial sequences of GABABR1, partial sequences of GABABR2, transmembrane sequences (and fluorescent tags), a cell line with stable and high expression of the recombinant protein is constructed by transfecting CHO cells or HEK293 cells, which can significantly improve the sensitivity and specificity in the detection of anti-GABABR antibody encephalitis, and can achieve the purpose of detecting antibodies by using the fixed CBA method or the live cell CBA method, and is applicable to the auxiliary diagnosis of autoimmune encephalitis in 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, specifically to a recombinant protein for detecting anti-GABABR antibody encephalitis by using the cell immunofluorescence method (CBA method) and related detection reagents containing the recombinant protein, belonging to the technical field of neuroimmunological diagnosis. Background Art

[0002] The γ-aminobutyric acid type B receptor (GABAB receptor, GABABR) is an important inhibitory G protein-coupled receptor (GPCR) in the central nervous system, and a functional receptor complex is formed by the heterodimerization of two subunits, GABABR1 and GABABR2. In recent years, studies have found that autoantibodies against GABABR (anti-GABABR antibodies) are closely related to various autoimmune neurological diseases, especially anti-GABABR antibody encephalitis, whose clinical manifestations include seizure, cognitive impairment, and mental and behavioral abnormalities, etc. Rapid and accurate detection of anti-GABABR antibodies in the serum or cerebrospinal fluid of patients 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 separately express a single subunit as antigens, such as single expression of GABABR1 or GABABR2. However, their antigen structures cannot mimic the natural receptor structure, easily resulting in insufficient exposure of antibody-binding epitopes, significantly reducing the detection sensitivity and increasing the false negative rate. (2) Using recombinant proteins that co-express GABABR1 and GABABR2 subunits to mimic the natural receptor heterodimer structure. However, when separately constructing independent vectors for co-transfection to express GABABR1 and GABABR2, since the independent expression systems of the two subunits are difficult to precisely regulate the expression ratio, it is easy to cause imbalance in the subunit expression levels in the cell population. At the same time, the formation efficiency of the heterodimer is affected by transfection conditions (such as plasmid ratio, transfection reagent, cell state, etc.), significantly increasing the uncontrollability of experimental variables. It can be seen that the co-expression of GABABR1 and GABABR2 subunits easily leads to an increase in batch-to-batch differences in antigen 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. This patent clearly states that the etiology of encephalitis can be determined by detecting autoantibodies against the GABAB receptor and its subtypes (including the B1 subunit and B2 subunit of GABAB) or GABAB receptor multimers (such as the B1 / B2 subunit heterodimer, the combination of B1a / b / c subtypes and the B2 subunit). Although this patent proposes a scheme using GABAB receptor multimers as detection targets, in practical applications, these heteromultimers composed of different subunits still face significant technical challenges during construction: the first is the uncontrollability of expression: the subunit ratio imbalance and assembly efficiency are greatly affected by transfection conditions; the second is the structural stability: it is difficult to ensure the integrity of the native conformation of the heterodimer; the third is the bottleneck of detection performance: the above factors will directly restrict the homogeneity of the antigen structure, thereby affecting the specificity and sensitivity of the detection of anti-GABABR antibody encephalitis.

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

[0006] The object 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 its detection in anti-GABABR antibody encephalitis can be significantly improved. At the same time, the present invention also provides a detection reagent containing this recombinant antigen, which is suitable for the auxiliary diagnosis of autoimmune encephalitis in cerebrospinal fluid / serum.

[0007] The present invention is achieved through the following technical solutions: A recombinant protein for detecting anti-GABABR antibody encephalitis, containing a partial sequence of GABABR1, a partial sequence of GABABR2, and a transmembrane sequence,

[0008] The partial sequence of GABABR1 is as shown in SEQ ID NO: 1,

[0009] The partial sequence of GABABR2 is as shown in SEQ ID NO: 2,

[0010] The transmembrane sequence is CD8a hinge.

[0011] It also includes: a first linker peptide connecting the partial sequence of GABABR1 and the partial sequence of GABABR2, and a second linker peptide connecting the partial sequence of GABABR2 and the transmembrane sequence.

[0012] The sequence of the first linker peptide is shown as SEQ ID NO: 4, and the sequence of the second linker peptide is shown as SEQ ID NO: 5.

[0013] The transmembrane sequence is linked to a fluorescent tag through a third linker peptide.

[0014] The fluorescent tag is mCherry.

[0015] The sequence of the third linker peptide is shown as SEQ ID NO: 7.

[0016] A detection reagent for anti-GABABR antibody encephalitis, the nucleic acid encoding the above recombinant protein is co-transfected into cells, and the obtained expressing cells are used as a detection reagent for anti-GABABR antibody encephalitis.

[0017] The host cells for the nucleic acid co-transfection include CHO cells or HEK293 cells.

[0018] Its detection form is the fixed CBA method or the live cell CBA method.

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

[0020] (1) The present invention selectively intercepts partial sequences in the open reading frames of GABABR1 and GABABR2, uses a linker peptide to fuse the selected sequences of the two subunits, and introduces a transmembrane domain, enabling the antigenic epitope to be correctly folded and expressed on the cell membrane surface, achieving the detection sensitivity of co-transfection of the GABABR1 and GABABR2 double subunits, and enabling the purpose of detecting antibodies by the fixed CBA method or the live cell CBA method.

[0021] (2) The present invention has specific truncated sequences of both GABABR1 and GABABR2, solving the problem of subunit-dependent antibody recognition where some patients' sera only recognize the GABABR1 or GABABR2 subunit, while another part depends on the co-expressed structure of both subunits, making the detection results more comprehensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the map of the lentiviral vector in the present invention.

[0023] Figure 2 It is the immunofluorescence image of the positive sample in Example 1 of the present invention.

[0024] Figure 3 It is the immunofluorescence image of the negative sample in Example 1 of the present invention.

[0025] Figure 4 It is the comparison diagram of the protein structure and the natural conformation in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The invention purpose, technical solution and beneficial effects of the present invention will be further described in detail below.

[0027] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the claimed invention. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0028] Through innovative fusion protein design, the present invention respectively intercepts partial sequences of GABABR1 and GABABR2, selects a linking sequence to fuse them, constructs a lentiviral expression vector, and then stably transfects CHO cells or HEK293 cells, and further screens monoclonal cell lines with stable high expression of recombinant proteins for subsequent detection.

[0029] The technical solution of the present invention can be specifically summarized as follows:

[0030] (I) Recombinant protein

[0031] The present invention forms a recombinant protein by selectively intercepting partial sequences of GABABR1 and GABABR2, and then recombinantly constructing them with a first linker peptide linker1, a second linker peptide linker2 and a transmembrane sequence respectively. As a diagnostic antigen for detecting anti-GABABR antibody encephalitis, this recombinant protein design not only retains the key antigenic epitopes of the natural receptor, but also achieves stable expression on the cell membrane surface through the linking sequence (i.e., the first linker peptide linker1, the second linker peptide linker2 and the transmembrane sequence), with a complete conformation.

[0032] Specifically, the partial sequence of GABABR1 comes from the 1-578 amino acid sequence of the N-terminal domain of the transcriptional isoform GABABR1, and its sequence is as shown in SEQ ID NO: 1 as follows:

[0033] MLLLLLLAPLFLRPPGAGGAQTPNATSEGCQIIHPPWEGGIRYRGLTRDQVKAINFLPVDYEIEYVCRGEREVVGPKVRKCLANGSWTDMDTPSRCVRICSKSYLTLENGKVFLTGGDLPALDGARVDFRCDPDFHLVGSSRSICSQGQWSTPKPHCQVNRTPHSERRAVYIGALFPMSGGWPGGQACQPAVEMALEDVNSRRDILPDYELKLIHHDSKCDPGQATKYLYELLYNDPIKIILMPGCSSVSTLVAEAARMWNLIVLSYGSSSPALSNRQRFPTFFRTHPSATLHNPTRVKLFEKWGWKKIATIQQTTEVFTSLDDLEERVKEAGIEITFRQSFFSDPAVPVKNLKRQDARIIVGLFYETEARKVFCEVYKERLFGKKYVWFLIGWYADNWFKIYDPSINCTVDEMTEAVEGHITTEIVMLNPANTRSISNMTSQEFVEKLTKRLKRHPEETGGFQEAPLAYDAIWALALALNKTSGGGGRSGVRLEDFNYNNQTITDQIYRAMNSSSFEGVSGHVVFDASGSRMAWTLIEQLQGGSYKKIGYYDSTKDDLSWSKTDKWIGGSPPADQTL。

[0034] The partial sequence of GABABR2 is the amino acid sequence from 26 to 469 of GABABR2, and its sequence is as shown in SEQ ID NO: 2 as follows:

[0035] LLLLPLLLPLAPGAWGWARGAPRPPPSSPPLSIMGLMPLTKEVAKGSIGRGVLPAVELAIEQIRNESLLRPYFLDLRLYDTECDNAKGLKAFYDAIKYGPNHLMVFGGVCPSVTSIIAESLQGWNLVQLSFAATTPVLADKKKYPYFFRTVPSDNAVNPAILKLLKHYQWKRVGTLTQDVQRFSEVRNDLTGVLYGEDIEISDTESFSNDPCTSVKKLKGNDVRIILGQFDQNMAAKVFCCAYEENMYGSKYQWIIPGWYEPSWWEQVHTEANSSRCLRKNLLAAMEGYIGVDFEPLSSKQIKTISGKTPQQYEREYNNKRSGVGPSKFHGYAYDGIWVIAKTLQRAMETLHASSRHQRIQDFNYTDHTLGRIILNAMNETNFFGVTGQVVFRNGERMGTIKFTQFQDSREVKVGEYNAVADTLEIINDTIRFQGSEPPKDKTI。

[0036] The transmembrane sequence is the CD8a hinge, that is, the amino acid sequence 183-206 of the hinge region of CD8a, and its sequence is as shown in SEQ ID NO: 3 as follows:

[0037] IYIWAPLAGTCGVLLLSLVITLYC。

[0038] The sequence of the first linker linker1 is as shown in SEQ ID NO: 4 as follows:

[0039] GSTGGGGSGGGGSGGGGSGAASR。

[0040] The sequence of the second linker linker2 is as shown in SEQ ID NO: 5 as follows:

[0041] ASGGGGSGGGGSSG。

[0042] Furthermore, the present invention forms a traceable fusion expression system by inserting a fluorescent tag into the vector framework of the above recombinant protein. For example, the fluorescent tag is connected to the transmembrane sequence of the recombinant vector through the third linker linker3 to obtain a recombinant protein with the structure of partial sequence of GABABR1-linker1-partial sequence of GABABR2-linker2-CD8a hinge-linker3-mCherry.

[0043] Specifically, the fluorescent label is mCherry, and its sequence is as shown in SEQ ID NO: 6 as follows:

[0044] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0045] The sequence of the third linker peptide linker3 is as shown in SEQ ID NO: 7 as follows:

[0046] AEAAAKEAAAKA.

[0047] (2) Detection reagent

[0048] The present invention uses the above recombinant protein to prepare related reagents for detecting anti-GABABR antibody encephalitis. For example, the purified recombinant protein is covalently coupled to fluorescently encoded microspheres to detect anti-GABABR antibody encephalitis in the fixed CBA method; or live cells expressing the recombinant protein are used to detect anti-GABABR antibody encephalitis in the live cell CBA method.

[0049] In a possible embodiment, to construct a lentiviral vector of the recombinant protein GABABR1-GABABR2-CD8a hinge-mCherry, the above partial sequences of GABABR1, partial sequences of GABABR2, the transmembrane sequence CD8a hinge, and the fluorescent label mCherry can be sequentially connected through the first linker peptide linker1, the second linker peptide linker2, and the third linker peptide linker3 to form, and the map of the lentiviral vector is as Figure 1 shown.

[0050] In a possible embodiment, to prepare a detection reagent for the above recombinant protein, the constructed lentiviral vector can be transfected into CHO cells or HEK293 cells, and monoclonal cells that stably and highly express the GABABR1-GABABR2-CD8a hinge-mCherry recombinant protein can be selected. 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. When they grow to a certain confluence, they can be used to prepare detection reagents for subsequent immunofluorescence experiments.

[0051] 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%.

[0052] 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.

[0053] The anti-GABABR antibody encephalitis immunofluorescence detection method of the present invention is as follows:

[0054] Prepare blood or cerebrospinal fluid samples with sample diluent containing the following:

[0055] Phosphate buffer (pH 7.0-7.4), BSA (2%);

[0056] 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;

[0057] Sample incubation: 37°C for 1 hour, washed 3 times with PBS;

[0058] 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;

[0059] Interpretation: The positive or negative status of the sample is determined under a microscope.

[0060] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0061] Embodiment 1:

[0062] 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.

[0063] Comparative Example 1:

[0064] This comparative example uses the full-length GABABR1 sequence (1-961aa) (see NM_001470.4) to express and detect anti-GABABR antibody encephalitis.

[0065] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfecting CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and fixing with paraformaldehyde after culturing for 48 hours.

[0066] Comparative Example 2:

[0067] This comparative example uses the full-length GABABR2 sequence (1-941aa) (see NM_005458.8) to express and detect anti-GABABR antibody encephalitis.

[0068] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfecting CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and fixing with paraformaldehyde after culturing for 48 hours.

[0069] Comparative Example 3:

[0070] This comparative example constructs lentiviral vectors using the full-length GABABR1 sequence (1-961aa) and the full-length GABABR2 sequence (1-941aa) respectively, and co-expresses to detect anti-GABABR antibody encephalitis.

[0071] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfecting CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and fixing with paraformaldehyde after culturing for 48 hours.

[0072] Comparative Example 4:

[0073] This comparative example uses the fusion expression of GABABR1 full-length sequence (1-961aa) + linker1 + GABABR2 full-length sequence (1-941aa) + linker2 + CD8a hinge (183-206aa) + linker3 + mCherry to detect anti-GABABR antibody encephalitis.

[0074] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfecting CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and fixing with paraformaldehyde after culturing for 48 hours.

[0075] Comparative Example 5:

[0076] 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.

[0077] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfect CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and after culturing for 48 hours, fix with paraformaldehyde.

[0078] Comparative Example 6:

[0079] 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.

[0080] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfect CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and after culturing for 48 hours, fix with paraformaldehyde.

[0081] Comparative Example 7:

[0082] 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.

[0083] By constructing a eukaryotic transient expression vector containing the above sequence, transiently transfect CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and after culturing for 48 hours, fix with paraformaldehyde.

[0084] Comparative Example 8:

[0085] 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.

[0086] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfect CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and after culturing for 48 hours, fix with paraformaldehyde.

[0087] Comparative Example 9:

[0088] In this comparative example, the fusion expression of partial sequence of GABABR1 (1 - 578aa) + linker1 + partial sequence of GABABR2 (26 - 300aa) + linker2 + CD8a hinge (183 - 206aa) + linker3 + mCherry was used to detect anti-GABABR antibody encephalitis.

[0089] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfect CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and after culturing for 48 hours, fix with paraformaldehyde.

[0090] Comparative Example 10:

[0091] In this comparative example, the fusion expression of partial sequence of GABABR1 (1 - 578aa) + linker1 + partial sequence of GABABR2 (26 - 717aa) + linker2 + CD8a hinge (183 - 206aa) + linker3 + mCherry was used to detect anti-GABABR antibody encephalitis.

[0092] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfect CHO cells or HEK293 cells with Lipofectamine™ 3000 transfection reagent, and after culturing for 48 hours, fix with paraformaldehyde.

[0093] Experimental Example:

[0094] Add the detection reagents prepared in Example 1 and Comparative Examples 1 to 10 above to positive samples clinically diagnosed with anti-GABABR antibody encephalitis respectively. According to the diagnostic criteria of the GABABR encephalitis expert consensus, 328 samples diagnosed with anti-GABABR antibody encephalitis were selected.

[0095] The samples included need to meet three conditions: A, B, and C:

[0096] A. One or more of the 6 main symptoms: (1) refractory epilepsy; (2) cognitive dysfunction; (3) mental and behavioral abnormalities; (4) disturbance of consciousness; (5) movement disorders; (6) autonomic dysfunction;

[0097] B. Positive for anti-GABABR antibody encephalitis: It is recommended to take the positive antibody result by cerebrospinal fluid CBA method as the standard. If only serum specimens are available for testing, in addition to the positive CBA result, TBA is also required for final confirmation, and a low-titer serum positive (1:10) has no diagnostic significance.

[0098] C. Reasonably exclude other etiologies.

[0099] The samples are serum and cerebrospinal fluid. Among them, the dilution ratios of serum are 1:10, 1:32, and 1:100; the cerebrospinal fluid is used undiluted without dilution.

[0100] The detection is carried out by immunofluorescence method, and the results are shown in Figure 2 and Table 1 below: Comparison of the detection rates of positive samples for GABABR antibody disease.

[0101] Figure 2 It is the immunofluorescence image of the positive sample in Example 1. In the figure, A is the red light of the GABABR1 and GABABR2 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 the red light and the green light, indicating that the sample is detected as positive.

[0102] Table 1: Comparison of the detection rates of positive samples for GABABR antibody disease

[0103]

[0104] From Figure 2 and Table 1, it can be seen that Example 1 using the method of the present invention can well detect positive samples of anti-GABABR antibody encephalitis, and when compared with the detection results of Comparative Example 1 to Comparative Example 10, it has better sensitivity at higher dilution multiples of serum. In Comparative Example 2, the antigen epitope is not contained and cannot be detected. In Comparative Examples 6-10, the selected sequence fragments of the fusion protein change the spatial conformation of the protein and thus cannot be detected.

[0105] The fixed cells prepared in Example 1, Comparative Example 1 to Comparative Example 10 above are respectively added with 24 cases of healthy human serum samples, 45 cases of sera of other patients without anti-GABABR antibody encephalitis, and 38 cases of cerebrospinal fluid samples.

[0106] The detection is carried out by immunofluorescence method, and the results are shown in Figure 3 and Table 2 below: Comparison of the detection rates of healthy human negative samples.

[0107] Figure 3 It is the immunofluorescence image of the negative sample in Example 1. In the figure, A is the red light of the GABABR1 and GABABR2 fusion protein itself, B is no green fluorescence after incubation of the negative sample with the secondary antibody, and C is the superimposed image of A and B, indicating that the sample is detected as negative.

[0108] Table 2: Comparison of the detection rates of negative samples in healthy individuals

[0109]

[0110] Furthermore, from Figure 3 and Table 2, it can be seen that Example 1 using the method of the present invention can detect negative samples well, and the specificity meets the requirements.

[0111] In summary, the method of the present invention can be used for the detection of GABABR autoantibodies by fusing and expressing specific partial sequences of GABABR1 and GABABR2. It has specificity and sensitivity, and its detection effect is better than those of Comparative Examples 1 to 10. Among Comparative Examples 1 to 10, Comparative Examples 1 and 2 are detection methods for separately expressing the full length of GABABR (GABABR1 or GABABR2 transcript), Comparative Examples 3 and 4 are methods for fusing and expressing the full lengths of GABABR1 and GABABR2, Comparative Examples 5 and 6 are for separately expressing the partial sequences of GABABR1 and GABABR2 in Example 1, and Comparative Examples 7 to 10 are for fusing and expressing partial sequences of GABABR1 and GABABR2, but their fusion sequences are different. Among them, GABABR1 is the same as that in Example 1, and the partial sequence of GABABR2 is longer or shorter than that in Example 1.

[0112] Furthermore, it should be noted that the present invention constructs a recombinant protein using specifically truncated GABABR1 and GABABR2 sequences, which can correctly fold the antigenic epitope and present the natural conformation (see Figure 4 , Figure 4 where A is the protein structure of Example 1, B is the natural conformation, and C is the comparison after overlapping A and B). Therefore, when used for the detection of anti-GABABR antibody encephalitis, it has significant specificity and sensitivity. However, due to different selected sequences in Comparative Examples 1 to 10, their conformational folding and expression levels are different, and they cannot provide a detection effect comparable to that of the present invention. In Comparative Examples 1 and 2, the full sequences of GABABR1 and GABABR2 are respectively selected, but their extracellular domains are incomplete compared to the natural conformation; in Comparative Example 3, co-expression of the full sequences of GABABR1 and GABABR2 is adopted, and the co-expression ratio is difficult to control and the expression level is poor; in Comparative Example 4, the full sequences of GABABR1 and GABABR2 are constructed into one expression vector, and the sequence is too long and the expression level is low; in Comparative Example 5, the extracellular domain of GABABR1 is adopted, and in Comparative Example 6, the extracellular domain of GABABR2 is adopted, and their conformations are incomplete compared to the natural conformation; in Comparative Examples 7 to 10, the extracellular domain of GABABR1 and different partial sequences of GABABR2 are adopted, and their structures are different from the natural conformation.

[0113] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modification or equivalent change made to the above embodiments 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: It consists of a sequentially connected partial GABABR1 sequence, a first linker peptide, a partial GABABR2 sequence, a second linker peptide, a transmembrane sequence, a third linker peptide, and a fluorescent tag. The partial GABABR1 sequence is as shown in SEQ ID NO:

1. The partial GABABR2 sequence is as shown in SEQ ID NO:

2. The transmembrane sequence is CD8a hinge, and its sequence is as shown in SEQ ID NO:

3. The sequence of the first linker peptide is as shown in SEQ ID NO:

4. The sequence of the second linker peptide is as shown in SEQ ID NO:

5. The fluorescent tag is mCherry, and its sequence is as shown in SEQ ID NO:

6. The sequence of the third linker peptide is as shown in SEQ ID NO:

7.

2. A detection reagent for anti-GABABR antibody encephalitis, characterized in that: The nucleic acid encoding the recombinant protein according to claim 1 is co-transfected into cells, and the resulting expressing cells are used as a detection reagent for anti-GABABR antibody encephalitis.

3. The detection reagent according to claim 2, characterized in that: The host cells for the nucleic acid co-transfection include CHO cells or HEK293 cells.

4. The detection reagent according to claim 2, wherein: The detection form is a fixed CBA method or a live cell CBA method.

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