Rat tex264 recombinant protein, recombinant vector, polyclonal antibody and application

Abstract

The application provides a rat TEX264 recombinant protein, a recombinant vector, a polyclonal antibody and application, and relates to the technical field of biological engineering.The amino acid sequence of the rat TEX264 recombinant protein is shown as SEQ ID NO.2.The application provides a preparation method of an anti-rat TEX264 polyclonal antibody, and a polyclonal antibody against rat TEX264 is prepared; the antibody can specifically recognize endogenous TEX264 protein of a rat, and is used for experiments such as ELISA, Western Blot, immunofluorescence analysis and the like.The application provides a material basis for studying the function of TEX264.The antibody of the application can detect TEX264 protein of human and mouse tissues in addition to recognizing and detecting rat TEX264, compared with a related antibody on the market which can only detect TEX264 protein of mouse and / or human tissue samples; the antibody can simultaneously detect TEX264 protein of rat, mouse and human tissue samples, and is suitable for various different detection methods such as Western Blot, immunohistochemistry and immunofluorescence, and has advantages in economic benefits.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a rat TEX264 recombinant protein, recombinant vector, polyclonal antibody, and its applications. Background Technology

[0002] Testis expressed 264 (TEX264) is a single-transmembrane protein composed of an N-terminal hydrophobic region, a Gyrl-like domain that inhibits topoisomerase, and a loosely structured C-terminus. As a major endoplasmic reticulum (ER) autophagy receptor, TEX264, independent of other ER receptors, can remodel ER subdomains into autophagosomes under nutritional stress, subsequently achieving ER metabolic cycling through fusion with lysosomes. During amino acid deprivation or ER stress (such as tunicamycin treatment), the conserved LIR motif at the C-terminus of TEX264 directly binds to the hydrophobic pocket of ATG8 family proteins (such as LC3B and GABARAP). The ATG8-containing insulatory membrane (IM) envelops the tubular region of the ER near the TEX264-positive ER junction, forming a double-membrane synaptic structure through the trans-interaction of TEX264 with ATG8 proteins. Multiple TEX264-ATG8 interacting pairs form a periodic arrangement along the ER-IM interface, driving IM expansion through progressive binding, much like zipper closure. The closed autophagosome fuses with the lysosome via the Rab7-STX17-SNAP29 complex, and the endoplasmic reticulum components are degraded by acidic hydrolases. This protein also participates in the repair of covalent DNA-protein crosslinks (DPCs) during DNA synthesis by bridging VCP / p97 to DPC sites, initiating the SPRTN-mediated DPC dissociation pathway. Recently, TEX264 has been identified as a cofactor of VCP / p97 ATPase, promoting the repair of covalently trapped TOP1 (DNA topoisomerase 1)-DNA crosslinks.

[0003] Currently, research on TEX264 focuses on its molecular mechanisms in endoplasmic reticulum autophagy and its role in DNA repair. However, the mechanisms by which TEX264 mediates membrane cleavage and triggers the rupture of related membrane structures have not yet been fully elucidated.

[0004] As one of the endoplasmic reticulum autophagy receptors, the mechanism of action of TEX264 in endoplasmic reticulum autophagy is not yet fully elucidated, especially since commercially available rat TEX264 polyclonal antibodies are still lacking. Existing antibodies against human and mouse TEX264 protein fragments do not readily recognize endogenous rat TEX264 protein. The reason for the need to recognize endogenous rat TEX264 protein in the current technology is that TEX264 mRNA expression was upregulated in the inventors' tail-suspension simulated weightlessness rat animal model. To further investigate the relevant mechanism of action, it is necessary to detect the TEX264 protein level in the rat model. The development of rat TEX264 antibodies will also facilitate research on TEX264 in other rat-based animal models. Summary of the Invention

[0005] To address the technical problems existing in the prior art, embodiments of the present invention provide a rat TEX264 recombinant protein, a recombinant vector, a polyclonal antibody, and its applications. The technical solution is as follows:

[0006] A rat TEX264 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] A recombinant vector expressing the rat TEX264 recombinant protein, the recombinant vector comprising a DNA sequence encoding the rat TEX264 recombinant protein, wherein the DNA sequence is shown in SEQ ID NO.3.

[0008] Optionally, the plasmid used to construct the recombinant vector is the pET-22b expression vector.

[0009] A method for preparing the rat TEX264 recombinant protein, characterized in that the method comprises:

[0010] 1) Transform the recombinant vector into competent cells to construct engineered bacteria carrying the recombinant vector;

[0011] 2) The engineered bacteria were induced with IPTG to express the rat TEX264 recombinant protein, which was then purified.

[0012] A polyclonal antibody against rat TEX264 protein, said polyclonal antibody being prepared by animal immunization using the recombinant rat TEX264 protein.

[0013] The method for preparing the polyclonal antibody against rat TEX264 protein includes:

[0014] (1) Preparation of the rat TEX264 recombinant protein;

[0015] (2) Use the rat TEX264 recombinant protein obtained in step (1) as an antigen to immunize animals;

[0016] (3) After immunizing the animals in step (2), the animal serum is obtained and purified to obtain the polyclonal antibody against rat TEX264 protein.

[0017] Optionally, the animal mentioned in step (2) is a rabbit.

[0018] Optionally, the immunization conditions described in step (2) are multiple subcutaneous injections on the back, a total of 4 immunizations, the first immunization is performed after emulsification with Freund's complete adjuvant, and the second, third and fourth immunizations are performed after emulsification with Freund's incomplete adjuvant at weeks 2, 4 and 6.

[0019] The application of the polyclonal antibody against rat TEX264 protein in a specific detection method for rat TEX264 protein, wherein the specific detection method refers to a detection method that uses a polyclonal antibody against rat TEX264 protein to identify rat TEX264 protein.

[0020] A specific detection method for TEX264 protein, comprising:

[0021] (1) Obtaining protein samples: The protein samples are rat TEX264 protein;

[0022] (2) The protein sample was transferred to a PVDF membrane after SDS-PAGE gel electrophoresis and blocked with PBST buffer at room temperature;

[0023] (3) The polyclonal antibody against rat TEX264 protein was diluted in PBST buffer to prepare a primary antibody working solution. The PVDF membrane from step (2) was immersed in the primary antibody working solution, incubated at room temperature, and washed.

[0024] (4) Add HRP-labeled secondary antibody, incubate at room temperature, and wash;

[0025] (5) Expose with luminescent liquid and observe the results.

[0026] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0027] This invention provides a method for preparing a polyclonal antibody against rat TEX264, which specifically recognizes the rat endogenous TEX264 protein and can be used in experiments such as ELISA, Western blotting, and immunofluorescence analysis. This invention provides a material basis for studying the function of TEX264.

[0028] In addition to detecting TEX264 in rats, the antibody of this invention can also detect TEX264 protein in human and mouse tissues. Compared with commercially available antibodies that can only detect TEX264 protein in mouse and / or human tissue samples, this antibody can simultaneously detect TEX264 protein in rat, mouse and human tissue samples, and is applicable to various detection methods such as Western blotting, immunohistochemistry and immunofluorescence, which has advantages in terms of economic benefits. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The image shows the SDS-PAGE electrophoresis results of low-level expression of pET22b-TEX264 recombinant plasmid protein. Lane 1 is the protein marker, lane 2 is the control, and lane 3 is the IPTG-induced group.

[0031] Figure 2 The image shows the SDS-PAGE electrophoresis results of the large-scale expression and purification of pET22b-TEX264 recombinant plasmid protein. Lane 1 is the protein marker, lane 2 is the supernatant of the bacterial culture after sonication, lane 3 is the bacterial culture precipitate after sonication, lane 4 is the protein sample eluted with 60mM imidazole, lane 5 is the protein sample eluted with 200mM imidazole, and lane 6 is the protein sample eluted with 500mM imidazole.

[0032] Figure 3 The image shows the SDS-PAGE electrophoresis results of concentrated pET22b-TEX264 recombinant plasmid protein, where M is the protein marker and lane 1 is the concentrated protein band.

[0033] Figure 4 Line graph for ELISA serum titer determination;

[0034] Figure 5 This is an SDS-PAGE image of the TEX264 antibody, with a purity of 98%.

[0035] Figure 6A and Figure 6B This is a Western blot validation image of the anti-rat TEX264 protein polyclonal antibody, in which... Figure 6A The image shows the Western blot (WB) validation results for detecting recombinant TEX264 protein with TEX264 polyclonal antibody. Figure 6BWestern blot (WB) validation results of TEX264 polyclonal antibody detection of endogenous TEX264 protein expression in human carotid intima, rat brain, and mouse brain tissue;

[0036] Figure 7 Immunofluorescence validation results of the detection of endogenous TEX264 protein expression in human carotid intima, rat brain tissue and mouse brain tissue using polyclonal antibody against rat TEX264 protein;

[0037] Figure 8 Immunohistochemical verification results of endogenous TEX264 protein expression in human carotid intima, rat brain tissue, and mouse brain tissue using polyclonal antibodies against rat TEX264 protein. Detailed Implementation

[0038] The technical solutions of the present invention will now be described with reference to the accompanying drawings and embodiments.

[0039] Since there are no commercially available anti-rat TEX264 antibodies in the existing technology, the technical problem to be solved by this invention is to overcome the current lack of anti-rat TEX264 antibodies that can be used for scientific research or application. This invention provides a method for preparing a polyclonal anti-rat TEX264 antibody, which specifically recognizes the rat endogenous TEX264 protein and can be used in experiments such as ELISA, Western Blot, and immunofluorescence analysis. This invention provides a material basis for studying the function of TEX264.

[0040] In this invention, based on the rat TEX264 sequence published by NCBI, its antigenicity was analyzed. The antigen encoded by the 41st to 220th aa (180 aa) of the 472-1011 bp gene sequence (540 bp in length) was selected. After codon optimization of the 472-1011 bp gene sequence, a gene fragment was synthesized and pET22b-TEX264 was expressed using a prokaryotic expression system. Rabbit anti-TEX264 serum polyclonal antibodies were prepared by purifying and concentrating the recombinant TEX264 protein. This invention fills the gap in the lack of commercial anti-rat TEX264 antibodies and provides basic data for research on TEX264 in rats. The specific detection method for rat TEX264 protein of the present invention has the following objectives: 1) Ensure antibody specificity: Ensure that the polyclonal antibody for rat TEX264 protein can specifically recognize TEX264 protein and avoid non-specific binding or cross-reaction; 2) Evaluate antibody applicability: In the present invention, the antibody can be used for Western Blot, immunohistochemistry and immunofluorescence detection; 3) Verify antibody reliability: Verify the reliability and reproducibility of the antibody under different experimental conditions through various experimental methods (Western Blot, immunohistochemistry and immunofluorescence); 4) Standardize the detection process.

[0041] Example 1: Optimization of partial nucleotide sequence of TEX264 and gene synthesis

[0042] The full-length sequence of rat TEX264 protein (NP_001007666.1) from NCBI was analyzed for secondary structure, hydrophilicity / hydrophobicity, and antigenicity. Nucleotide sequence 472-1011 bp, encoding amino acids 41-220aa as shown in SEQ ID NO.1, was selected as the immunogen. The corresponding amino acid sequence of the rat TEX264 polypeptide is shown in SEQ ID NO.2.

[0043] To improve protein expression levels in prokaryotes, the nucleotide sequence 472-1011 bp shown in SEQ ID NO.1 was codon-optimized, and the optimized sequence is shown in SEQ ID NO.3. Gene fragment synthesis was then performed. The gene fragment synthesis was completed by Qingke Biotechnology Co., Ltd.

[0044] The sequence of SEQ ID NO.1 is as follows:

[0045] CCCCAATCCGCAACATAACTCTGGCCTACAAGTTCCATGTGGGGCCCTATGGTGACACTGGACAGCTTTTCACGGAGAGCTGCAGCATCTCTCCCAAGCTCCGCTCCATCGCTGTCTACTATGACAACCCCCAGACGGTGCCTCCCGAGAAGTGCCGCTGTGCAGTGGGCAGCATCCTGAGTGAGGGGGAGGAGTCGCCTTCACCTGAGCTCATCCACCTCTACCAGAAGTTTGGCTTCAAGATTTTCTCCTTTCCAGCACCTAGCCACGTGGTCACGACTACCTTCCCTTACACCACCCCCATATCCATCTGGCTGGCCGCCCGCCGAGTCCATCCTGCTTTGGATGTCTACATCAAGGAGCGGAAGCTGTGTGCTCACCCTCGGCTGGAGATCTACCAGCAAGACCAGATCCACTTCATGTGCCCACTGGCACGGCAAGGAGACTTCTATGTGCCAGAGGTGAAGGAGACAGAGCGGAAATGCTGGGAGCTTGTGGAGGCCACTGACACCCAGACGGATGGCACAGGAGCTGATACAA;

[0046] The sequence of SEQ ID NO.2 is:

[0047] PPIRNITLAYKFHVGPYGDTGQLFTESCSISPKLRSIAVYYDNPQTVPPEKCRCAVGSILSEGEESPSPELIHLYQKFGFKIFSFPAPSHVVTTTFPYTTPISIWLAARRVHPALDVYIKERKLCAHPRLEIYQQDQIHFMCPLARQGDFYVPEVKETERKCWELVEATDTQTDGTGADT;

[0048] The sequence of SEQ ID NO.3 is:

[0049] CTTAACGGATCGGTTGTTGGTGGTGGTGGTAGTGGTGGCGGTGGTTCACCGCCTATTCGCAATATTACCCTGGCATACAAATTTCATGTTGGTCCGTATGGTGATACCGGTCAGCTGTTTACCGAAAGCTGTAGCATTAGCCCGAAACTGCGTAGCATTGCAGTGTATTATGATAATCCGCAGACCGTTCCGCCTGAAAAATGTCGTTGTGCAGTTGGTAGCATTCTGAGCGAAGGTGAAGAAAGCCCGAGTCCGGAACTGATTCATCTGTATCAGAAATTCGGCTTCAAAATCTTTAGCTTTCCGGCACCGAGCCATGTTGTTACCACCACCTTTCCGTATACCACACCGATTAGCATTTGGCTGGCAGCACGTCGTGTTCATCCGGCACTGGATGTTTATATCAAAGAACGTAAACTGTGTGCACATCCGCGTCTGGAAATTTATCAGCAGGATCAGATTCACTTTATGTGTCCGCTGGCACGTCAGGGTGATTTTTATGTTCCGGAAGTTAAAGAAACCGAACGCAAATGTTGGGAATTAGTTGAAGCAACCGATACACAGACCGATGGTACAGGTGCAGATACCCATCACCACCATCATCATTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCA。

[0050] Example 2 Construction of pET22b-TEX264 Recombinant Vector and Preparation of Engineered Bacteria

[0051] The correctly synthesized TEX264 gene fragment was directionally cloned into the pET-22b expression vector using T4 ligase to construct the recombinant plasmid pET22b-TEX264. The construction of the recombinant plasmid pET22b-TEX264 was completed by Beijing Ruijinte Biotechnology Co., Ltd.

[0052] Transformation experiments were then performed. Frozen BL21(DE3) competent cells were thawed on ice, and 0.5–1 μg of recombinant plasmid was added and gently mixed. The cells were then incubated on ice for 30 min. After heat shock at 42°C for 90 s, the cells were immediately incubated on ice for 1–2 min. Preheated 800 μL of LB liquid medium was added, and the cells were incubated at 37°C with shaking at 158 ​​rpm for 50 min. After centrifugation (6000 rpm, 4 min), 650 μL of supernatant was removed, and the remaining 150 μL of bacterial culture was thoroughly resuspended and evenly spread onto LB solid medium containing ampicillin (60 µg / ml). Finally, the plates were inverted and incubated at 37°C for 12–16 h. Positive single colony clones containing the recombinant plasmid were obtained through antibiotic resistance screening.

[0053] Example 3 Expression of recombinant TEX264 protein in rats

[0054] 1. Small quantity expression:

[0055] The correctly sequenced pET-22b-TEX264 recombinant plasmid vector was transformed into BL21(DE3) competent cells. Single colonies were picked and cultured in 3 ml of LB liquid medium containing ampicillin (60 µg / ml) at 37°C and 200 rpm. After OD600 = 0.6, a portion of the bacterial culture was transferred to a new tube as a negative control. The remaining bacterial culture was induced with 1 mM IPTG and cultured at 18°C ​​with shaking for 3 h.

[0056] Take 0.15 mL of each of the two bacterial cultures, centrifuge at 12000×g for 2 min, collect the bacterial precipitate, blow it away with 40 μl of 10 mM Tris-HCl (pH 8.0) solution, resuspend it in 40 μL of 1×loading buffer for lysis, boil at 100℃ for 5 min, and detect whether the protein is expressed by SDS-PAGE electrophoresis.

[0057] Experimental results:

[0058] Small expression results, such as Figure 1 As shown. From Figure 1 As can be seen, the IPTG-induced group (lane 3) exhibits a specific expression band at a relative molecular mass of approximately 32 kDa. The experimental results demonstrate that IPTG can indeed induce the expression of the target protein.

[0059] 2. Extensive expression

[0060] 10 μl of the activated bacterial culture from step 1 was inoculated into 10 ml of the corresponding antibiotic-resistant LB liquid medium (containing ampicillin, ampicillin concentration 60 µg / ml) and cultured overnight at 37°C with shaking. The cultured bacterial culture was then transferred to 700 ml of the corresponding antibiotic-resistant LB liquid medium and cultured at 37°C and 200 rpm until OD600 = 0.6. 1 mM IPTG was added for induction, and the culture was continued at 18°C ​​with shaking for 4 h. Afterwards, the culture was centrifuged at 6000 rpm / min for 3 min, the bacterial precipitate was collected, and resuspended in a 20 mM Tris-HCl, 0.5 M NaCl (pH 8.0) buffer.

[0061] Bacterial cells were disrupted by sonication (power controlled at 300W, sonication for 4 seconds, pause for 4 seconds, sonication repeated 99 times). Centrifuged at 16000 rpm and 4℃ for 50 min, and the supernatant and precipitate were collected separately. The supernatant and precipitate were resuspended in 40 μl of 1XSDS-PAGE loading buffer, and after boiling in a water bath for 5 min, 10 μl was taken for protein electrophoresis. The remaining supernatant and precipitate were stored at 4℃ for later use.

[0062] Experimental results:

[0063] The results of the identification of high expression of pET-22b-TEX264 recombinant plasmid protein are as follows: Figure 2 As shown. From Figure 2 As can be seen, the supernatant of sample pET-22b-TEX264 (lane 2) has a specific expression band at a relative molecular mass of approximately 32 kDa, which is more specific than the band in the pET-22b-TEX264 precipitate (lane 3). This indicates that the recombinant plasmid was successfully expressed and mainly exists in the supernatant.

[0064] Example 4: Purification and Concentration Experiment of Recombinant Rat TEX264 Protein

[0065] The supernatant containing TEX264 protein was filtered through a 0.45 µm filter to remove impurities. The Ni-NTA affinity chromatography column (MCE, model hy-k0221) was equilibrated with a buffer containing 20 mM Tris-HCl and 0.5 M NaCl (pH 8.0) at a flow rate of 1 mL / min for pretreatment. The filtered protein sample was loaded onto the column at the same flow rate. After TEX264 protein specifically bound to nickel ions, the column was washed with equilibration buffer until the Bradford assay (G250 reagent) showed no protein residue in the eluent (the blue color remained stable). A gradient of imidazole buffers (20 mM, 60 mM, 200 mM, 500 mM) was used for stepwise elution, collecting each eluent until the G250 assay showed no further color development. After elution, the column was washed sequentially with 3 column volumes of deionized water, and the column was sealed with 20% ethanol for preservation.

[0066] The collected eluted fractions at various concentrations were analyzed by SDS-PAGE electrophoresis to screen for high-purity target protein fractions. These fractions were then dialyzed into PBS buffer (pH 7.4) and stored at -80°C for later use. The dialysis procedure is as follows:

[0067] (1) Pretreatment of dialysis bags (Fusheng A1999764): New dialysis bags are treated with 10% glycerol to remove sulfides, heavy metals, and some impurities with ultraviolet absorption. The dialysis bags are cut into 100-120 mm segments, boiled in 50% ethanol for 1 hour, then washed sequentially with 50% ethanol, 0.01 mol / L sodium bicarbonate, and 0.001 mol / L EDTA solution, and finally rinsed 3-5 times with distilled water. One end of the dialysis bag is clamped with a dialysis bag clamp, and the other end is filled with water. Slight pressure is applied with a finger to check for leaks. The treated dialysis bags are stored in distilled water for later use.

[0068] (2) Subsequently, 5 mL of the supernatant containing TEX264 protein was taken, and an equal volume of saturated ammonium sulfate was added. The mixture was allowed to stand for 15 minutes to precipitate the protein. The reversibility of the precipitation was verified by a reconstitution experiment. Finally, the saline protein solution was injected into a dialysis bag, suspended in distilled water, and the Cl in the beaker solution was measured at intervals. - (Silver nitrate method) and protein leaching (biuret reaction), change the water every 20 minutes until Cl - Completely remove the protein, and record any protein precipitation in the bag after dialysis.

[0069] Experimental results:

[0070] SDS-PAGE electrophoresis was used to identify the protein purification effect, such as... Figure 2 As shown in the diagram. Lane 4 contains protein samples eluted with 60 mM imidazole, lane 5 contains protein samples eluted with 200 mM imidazole, and lane 6 contains protein samples eluted with 500 mM imidazole. From Figure 2 As can be seen in lanes 4 to 6, the protein bands eluted with 200mM imidazole are relatively simple.

[0071] The results of specific bands after protein concentration can be found in [reference needed]. Figure 3 .from Figure 3 As can be seen, a single bright band was obtained after concentration and purification, indicating that high-purity recombinant protein was obtained after purification.

[0072] Example 5: Preparation of polyclonal antibodies against rat TEX264 protein

[0073] 1. Immunization of New Zealand White Rabbits

[0074] Initial immunization: New Zealand White rabbits weighing approximately 2.0 kg were selected. 500 µg of the TEX264 recombinant protein prepared in Example 4 was used as the immunogen, diluted to 400 µl with physiological saline, and then an equal volume of Freund's adjuvant was added and mixed thoroughly to form an oil emulsion. The rabbits were immunized by subcutaneous injection in the back at 8 injection sites.

[0075] Negative control group: A small amount of pre-immunization serum from healthy white rabbits was used as a blank control.

[0076] The immunization cycle was 49 days, with a total of 4 immunizations. The first immunization used Freund's complete adjuvant (purchased from MCE, model HY-153808) for emulsification. The second, third, and fourth immunizations were performed at weeks 2, 4, and 6 using Freund's incomplete adjuvant (purchased from MCE, model HY-153808A). The immunization dose was 500 µg. Blood samples were collected one week after the third immunization, and a large volume of blood was collected one week after the fourth immunization. Collected blood was incubated at 37°C for 2 hours to inactivate the serum, then incubated overnight at 4°C to release the serum. The serum was collected after two centrifugations at 5000 rpm / min for 10 minutes.

[0077] 2. ELISA detection of TEX264 polyclonal antibody titer

[0078] TEX264 protein was diluted to 2 µg / ml. Using carbonate buffer as the coating medium, 100 µl of the coated protein was added to each well of a 96-well plate and incubated overnight at 4°C. The next day, each well was washed three times with PBST buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.1% (v / v) Tween-20), then 200 µl of BSA was added, and the plate was blocked at 37°C for 1 hour. The plate was then washed three times again with PBST buffer.

[0079] Negative controls (using blank serum as a negative control) and post-immunization serum can be serially diluted at ratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000.

[0080] Add 100 µl of each sample to a pre-coated 96-well plate, repeating the process three times per sample, and incubate at 37°C for 1 h. After washing three times with PBST buffer, add 200 µL of HRP-labeled goat anti-rabbit IgG (Sino Biological, SSA004) (1:50000) to each well to label the primary antibody binding to the antigen (i.e., the TEX264 polyclonal antibody of this invention), and incubate at 37°C for 40 min. After washing three times with PBST buffer and twice with ultrapure water, add 100 µl of TMB chromogenic solution (Beyotime, P0209), and incubate in the dark at room temperature for 20 min. Finally, add 100 µl of hydrochloric acid to terminate the reaction. The absorbance (A450) of each well at 450 nm is then measured using a microplate reader. The highest dilution factor of the diluted antiserum is determined by reading the absorbance at 450 nm when it is greater than 2.1 times that of the pre-immune serum. That is, the highest dilution factor is defined as: titer = OD of antiserum / OD of pre-immune serum ≥ 2.1.

[0081] Experimental results:

[0082] The test results of potency are as follows Figure 4 As shown. From Figure 4 As can be seen, the titer of the antiserum (polyclonal antibody) is approximately 128K. This indicates that a polyclonal antibody against rat TEX264 protein was successfully prepared, and that the antibody has a high titer.

[0083] 3. Antibody purification and purity detection: 10 mg of TEX264 protein was dialyzed against 20 mM sodium bicarbonate buffer for 16 h, centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected and coupled with cyanogen bromide activated column material (Dalian Jingjian Biotechnology Co., Ltd., 09250) overnight at 4 °C. The column bed was washed sequentially with 20 mM sodium bicarbonate and Tris buffer, blocked, and stored in NaCl-sodium azide buffer (TSA buffer (pH=8), REGETE B0091). The coupling efficiency was verified by SDS-PAGE.

[0084] After equilibrating the antigen column, the antiserum sample prepared in step 1 was centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected and filtered through a 0.45 µm filter membrane. The pretreated antiserum sample was mixed with sodium acetate buffer at a ratio of 1:4 and loaded at 0.5 mL / min. The column was washed with sodium acetate buffer (0.2 mol / L sodium acetate and 0.0002 mol / L sodium hydroxide) until the G250 test was colorless. The column bed was washed with glacial acetic acid elution buffer (0.1 mol / L glacial acetic acid and 0.1 mol / L sodium acetate), and the elution peak was collected. The pH of the elution peak was quickly adjusted to neutral with saturated sodium carbonate. The eluent was concentrated to the original volume by ultrafiltration, centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected and temporarily stored at 4 °C for SDS-PAGE analysis. Purity was analyzed by SDS-PAGE Coomassie Brilliant Blue staining and Bandscan software. The final antibody was aliquoted and stored at -20 °C.

[0085] Experimental results:

[0086] The results of specific bands after purification of polyclonal antibodies can be found in [reference needed]. Figure 5 .from Figure 5 As can be seen, a single bright band was obtained after antibody purification, and the purity analysis result was 98%, indicating that a high-purity polyclonal antibody was obtained after purification.

[0087] Example 6 Specificity detection of TEX264 protein by anti-rat TEX264 protein polyclonal antibody

[0088] 1. Sample preparation

[0089] 1) Protein samples: Take 5, 10, 20 and 40 ng of rat TEX264 recombinant protein samples; take 0.1g of human carotid intima tissue, rat brain tissue and mouse brain tissue, add 1mL of protein lysis buffer (Solepro, R0020), homogenize with a tissue homogenizer, lyse at 4℃ for 30min, centrifuge at 12000rpm for 10min and take the supernatant. After BCA protein quantification, store at -80℃.

[0090] 2) Paraffin sections: Human carotid intima tissue, rat brain tissue and mouse brain tissue were fixed in 4% paraformaldehyde for 4 hours, and then successively immersed in a gradient of alcohol (50%→70%→80%→90%→95%→100%) for dehydration. The tissues were then successively immersed in xylene for 30 minutes for clearing treatment. After being immersed in paraffin, they were embedded in a mold to form a paraffin block, and 5μm thick sections were cut and preserved.

[0091] 2. Specific detection

[0092] 1) Immunoblotting:

[0093] Protein samples were separated by 12% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane under constant current of 60 mA for 80 min. Immediately after transfer, the membrane was blocked with PBST buffer at room temperature for 2 h.

[0094] The membrane was immersed in the working solution of the primary antibody prepared with the polyclonal antibody against rat TEX264 protein obtained in Example 5 (antibody diluted 1:1000 in PBST) and incubated at room temperature for 1 h. It was then washed four times with PBST for 10 min each time. HRP-labeled secondary antibody (purchased from Affinity, model S0001) was added, and the membrane was incubated at room temperature for 1 h. It was then washed four more times with PBST for 10 min each time. Development was performed using ECL chemiluminescence.

[0095] Experimental results:

[0096] The experimental results of the TEX264 recombinant protein sample of this invention are as follows: Figure 6A As shown. From Figure 6A As can be seen, when immunized rabbit positive serum was used as the primary antibody, a specific single band of recombinant TEX264 protein was detected at 32 kDa. The Western blot (WB) values ​​were consistent with the SDS-PAGE values, indicating that quality control was passed. TEX264 was also detected in human carotid intima, rat brain tissue, and mouse brain tissue. Figure 6B As shown. From Figure 6B As can be seen, when immunized rabbit positive serum was used as the primary antibody, a distinct specific band was detected at 32 kDa in human carotid intima, rat brain tissue, and mouse brain tissue. This indicates that the polyclonal antibody against rat TEX264 protein provided by this invention can specifically recognize rat TEX264 protein, as well as human and mouse TEX264 protein. This polyclonal antibody has high specificity and multi-gene reactivity.

[0097] 2) Immunofluorescence:

[0098] First, paraffin sections were dewaxed using a xylene gradient and then hydrated to distilled water using an ethanol gradient. Antigen retrieval was then performed using citric acid (pH 6.0) or EDTA (pH 9.0) buffer, via microwave heating on high for 15 minutes in stages or pressure cooking (3 minutes of steam). After retrieval, the sections were allowed to cool naturally and washed three times with PBS. The sections were permeabilized with Triton X-100 for 30 minutes, blocked with 1% BSA for 30 minutes, and then incubated overnight at 4°C in a humidified chamber with diluted polyclonal antibody against rat TEX264 protein (1:300 dilution). The next day, after washing with PBS, the sections were incubated for 50 minutes at room temperature in the dark with the corresponding FITC-labeled goat anti-rabbit secondary antibody (Proteintech, SA00003-2). After DAPI staining for 10 minutes to label cell nuclei, the sections were treated with an autofluorescence quencher (Beijing Pulilai Gene Technology Co., Ltd., C1212) for 5 minutes to eliminate background interference. Finally, the slides were thoroughly washed with PBS, mounted with an anti-fluorescence quenching mounting medium (Beijing Solarbio Science & Technology Co., Ltd., S2100), and stored at 4°C protected from light. During result interpretation, DAPI showed blue cell nuclei, and positive signals appeared green based on fluorescein labeling.

[0099] Experimental results:

[0100] Experimental results are as follows Figure 7 As shown. From Figure 7 As can be seen, when immunized rabbit positive serum was used as the primary antibody, green-labeled TEX264 protein signals and blue-labeled cell nuclear signals were detected in the human carotid intima, rat brain tissue, and mouse brain tissue. This indicates that the polyclonal antibody against rat TEX264 protein provided by this invention can be used for immunofluorescence detection and has multi-genus reactivity.

[0101] 3) Immunohistochemistry:

[0102] First, paraffin sections were dewaxed using a xylene gradient and then hydrated to distilled water using an ethanol gradient. Antigen retrieval was then performed using citric acid (pH 6.0) or EDTA (pH 9.0) buffer, via microwave heating on high for 15 minutes in stages or pressure cooking (3 minutes of steam). After retrieval, the sections were allowed to cool naturally and washed three times with PBS. To eliminate background interference, endogenous peroxidase was blocked for 25 minutes with 3% hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd., 10011218). After washing with PBS, the sections were blocked with goat serum (Beyotime Biotechnology, C0265) at room temperature for 15 minutes. A diluted primary antibody (polyclonal antibody against rat TEX264 protein, dilution ratio 1:300) was added and incubated overnight in a humidified chamber at 4°C. The next day, after washing with PBS, HRP-labeled secondary antibody (Zhongshan Jinqiao, ZB-2306) was added and incubated at room temperature for 50 minutes. DAB staining solution (Zhongshan Jinqiao, ZLI-9018) was used to control the staining time under a microscope (brownish-yellow indicates a positive result), and the reaction was terminated with tap water. Hematoxylin (Zhuhai Beso Biotechnology Co., Ltd., BA4041) was used to counterstain cell nuclei for 5-10 minutes. After differentiation with hydrochloric acid-ethanol and ammonia-based blue reversion, sections were dehydrated and cleared using a gradient of alcohol and xylene, and mounted with neutral resin. Final interpretation: cell nuclei appeared blue (hematoxylin staining), and positive signals appeared brownish-yellow (DAB staining).

[0103] Experimental results:

[0104] Experimental results are as follows Figure 8 As shown. From Figure 8 As can be seen, when immunized rabbit positive serum was used as the primary antibody, brown-labeled TEX264 protein signals and blue-labeled cell nuclear signals were detected in the human carotid intima, rat brain tissue, and mouse brain tissue. This indicates that the polyclonal antibody against rat TEX264 protein provided by this invention can be used for immunohistochemical detection and has multi-gene reactivity.

[0105] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A polyclonal antibody against rat TEX264 protein, characterized in that, The polyclonal antibody was prepared by animal immunization with rat TEX264 recombinant protein. The amino acid sequence of the rat TEX264 recombinant protein is shown in SEQ ID NO. 2; A method for preparing the rat TEX264 recombinant protein, the method comprising: 1) Transform the recombinant vector expressing the rat TEX264 recombinant protein into competent cells to construct engineered bacteria with the recombinant vector; 2) The engineered bacteria were induced with IPTG to express the rat TEX264 recombinant protein, which was then purified. The recombinant vector contains a DNA sequence encoding the rat TEX264 recombinant protein, wherein the DNA sequence is shown in SEQ ID NO. 3; The plasmid used to construct the recombinant vector was the pET-22b expression vector.

2. The method for preparing the polyclonal antibody against rat TEX264 protein according to claim 1, characterized in that, include: (1) Preparation of the rat TEX264 recombinant protein according to claim 1; (2) Use the rat TEX264 recombinant protein obtained in step (1) as an antigen to immunize animals; (3) After immunizing the animals in step (2), the animal serum is obtained and purified to obtain the polyclonal antibody against rat TEX264 protein.

3. The preparation method according to claim 2, characterized in that, The animal mentioned in step (2) is a rabbit.

4. The preparation method according to claim 2, characterized in that, The immunization conditions described in step (2) are multiple subcutaneous injections on the back, a total of 4 immunizations, the first immunization is performed after emulsification with Freund's complete adjuvant, and the second, third and fourth immunizations are performed after emulsification with Freund's incomplete adjuvant at weeks 2, 4 and 6.

5. The application of the polyclonal antibody against rat TEX264 protein according to claim 1 in a method for the specific detection of rat TEX264 protein, wherein the application is for non-disease diagnosis or treatment purposes, and wherein the specific detection method refers to a detection method that uses a polyclonal antibody against rat TEX264 protein to identify rat TEX264 protein.

6. A specific detection method for TEX264 protein, characterized in that, The specific detection methods are for non-disease diagnosis or treatment purposes, including: (1) Obtaining a protein sample: The protein sample is TEX264 protein; (2) The protein sample was transferred to a PVDF membrane after SDS-PAGE gel electrophoresis and blocked with PBST buffer at room temperature; (3) The polyclonal antibody against rat TEX264 protein according to claim 1 is diluted in PBST buffer to prepare a primary antibody working solution. The PVDF membrane from step (2) is immersed in the primary antibody working solution, incubated at room temperature, and washed. (4) Add HRP-labeled secondary antibody, incubate at room temperature, and wash; (5) Expose with luminescent liquid and observe the results.

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