Rat TEX264 recombinant protein, recombinant vector, polyclonal antibody and application

Abstract

The invention provides a rat TEX264 recombinant protein, a recombinant vector, a polyclonal antibody and application, and relates to the technical field of bioengineering. The amino acid sequence of the rat TEX264 recombinant protein is as shown in SEQ ID NO. 2. The invention provides a preparation method of an anti-rat TEX264 polyclonal antibody, the anti-rat TEX264 polyclonal antibody is prepared, and the antibody can specifically recognize rat endogenous TEX264 protein and is used for experiments such as ELISA (enzyme-linked immuno sorbent assay), Western Blot and immunofluorescence analysis. The invention provides a material basis for researching the function of TEX264. The antibody provided by the invention not only can identify TEX264 detection of rats, but also can detect TEX264 proteins of human and mouse tissues, and compared with related antibodies in the market only can detect TEX264 proteins of mice and (or) human tissue specimens, the antibody provided by the invention can simultaneously detect TEX264 proteins of rats, mice and human tissue specimens, and can be used for detecting the TEX264 proteins of the rats, mice and human tissue specimens. And the method is suitable for various different detection methods such as Western Blot, immunohistochemistry and immunofluorescence, and has advantages in the aspect of economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a rat TEX264 recombinant protein, a recombinant vector, a polyclonal antibody and applications thereof. Background Art

[0002] Testis expressed 264 (TEX264) is a single-pass transmembrane protein composed of an N-terminal hydrophobic region, a Gyrl (gyrase-like) domain and a loosely structured C-terminal. As the main endoplasmic reticulum autophagy receptor, TEX264 can reshape the endoplasmic reticulum subdomain into autophagosomes under nutrient stress conditions without relying on other endoplasmic reticulum autophagy receptors, and then achieve the endoplasmic reticulum metabolic cycle 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, GABARAP). The isolation membrane (IM) containing ATG8 wraps the endoplasmic reticulum tubular region near the three-way junction of TEX264-positive endoplasmic reticulum, and forms a double-membrane synaptic structure through the trans interaction between TEX264 and ATG8 proteins. Multiple TEX264-ATG8 interaction pairs form a periodic arrangement along the ER-IM interface, driving the expansion of IM through progressive binding, similar to the closing of a zipper. The closed autophagosome fuses with lysosomes through the Rab7-STX17-SNAP29 complex, and the endoplasmic reticulum components are degraded by acid hydrolases. This protein also participates in the repair of covalent DNA-protein crosslinks (DPCs) during DNA synthesis by bridging VCP / p97 to the DPCs site and initiating the SPRTN-mediated DPCs dissociation pathway. Recently, TEX264 was identified as a cofactor of the VCP / p97 ATPase, promoting the repair of covalently trapped TOP1 (DNA topoisomerase 1)-DNA crosslinks.

[0003] Currently, the research on TEX264 focuses on the molecular mechanism in the process of endoplasmic reticulum autophagy and its role in DNA repair. However, the mechanism of TEX264-mediated membrane fission and the associated membrane structure lysis triggered by it have not been fully elucidated.

[0004] As one of the autophagy receptors of the endoplasmic reticulum, the mechanism of TEX264 in the process of endoplasmic reticulum autophagy has not been fully elucidated, especially the lack of a commercial polyclonal antibody against rat TEX264. The existing antibodies with immunogens being human and mouse TEX264 protein fragments cannot well recognize the endogenous rat TEX264 protein. The reason for the need to recognize the endogenous rat TEX264 protein in the prior art is as follows: in the animal model of tail-suspended simulated weightlessness rats established by the present inventor, it was found that the expression of TEX264 mRNA was up-regulated. To further study the related mechanism of action, it is necessary to detect the protein level of TEX264 in the rat model. The development of rat TEX264 antibody is also helpful for the study of TEX264 in other rat-based animal models. Summary of the Invention

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

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

[0007] A recombinant vector expressing the recombinant protein of rat TEX264 as described above, wherein the recombinant vector contains a DNA sequence encoding the recombinant protein of rat TEX264, and the DNA sequence is as shown in SEQ ID NO.3.

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

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

[0010] 1) Transforming the recombinant vector into competent cells to construct an engineered bacterium carrying the recombinant vector;

[0011] 2) Inducing the engineered bacterium with IPTG to express the recombinant protein of rat TEX264 as described above and purifying it.

[0012] A polyclonal antibody against rat TEX264 protein, which is obtained by immunizing animals with the recombinant protein of rat TEX264 as described above.

[0013] The preparation method of the polyclonal antibody against rat TEX264 protein as described above comprises:

[0014] (1) Preparing the recombinant protein of rat TEX264 as described above;

[0015] (2) Immunize an animal with the rat TEX264 recombinant protein prepared in step (1) as an antigen;

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

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

[0018] Optionally, the immunization conditions in step (2) are multiple subcutaneous injections in the back, with a total of 4 immunizations. Use Freund's complete adjuvant for emulsification for the first immunization, and use Freund's incomplete adjuvant for emulsification for the second, third, and fourth immunizations 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 using the polyclonal antibody against rat TEX264 protein to recognize rat TEX264 protein.

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

[0021] (1) Obtain a protein sample: The protein sample is rat TEX264 protein;

[0022] (2) After subjecting the protein sample to SDS-PAGE gel electrophoresis, transfer it to a PVDF membrane and block it at room temperature with PBST buffer;

[0023] (3) Dilute the polyclonal antibody against rat TEX264 protein in PBST buffer to prepare a working solution of the primary antibody, immerse the PVDF membrane in step (2) in the working solution of the primary antibody, incubate at room temperature, and wash;

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

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

[0026] The beneficial effects brought by the technical solution provided by the embodiments of the present invention at least include:

[0027] The present invention provides a preparation method of a polyclonal antibody against rat TEX264, and prepares a polyclonal antibody against rat TEX264. This antibody can specifically recognize rat endogenous TEX264 protein and is used in experiments such as ELISA, Western Blot, and immunofluorescence analysis. The present invention provides a material basis for studying the function of TEX264.

[0028] In addition to being able to detect TEX264 in rats, the antibody of the present invention can also detect TEX264 protein in human and mouse tissues. Compared with the related antibodies on the market that can only detect TEX264 protein in mouse and / or human tissue specimens, this antibody can detect TEX264 protein in rat, mouse, and human tissue specimens simultaneously, and is applicable to various detection methods such as Western Blot, immunohistochemistry, and immunofluorescence, having advantages in terms of economic benefits. Brief Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0030] Figure 1 It is the SDS-PAGE electrophoresis result diagram of the small-scale expression of the pET22b-TEX264 recombinant plasmid protein. Among them, lane 1 is the protein Marker, lane 2 is the control, and lane 3 is the IPTG induction group;

[0031] Figure 2 It is the SDS-PAGE electrophoresis result diagram of the large-scale expression and purification of the pET22b-TEX264 recombinant plasmid protein. Lane 1 is the protein Marker, lane 2 is the supernatant of the bacterial solution after sonication, lane 3 is the precipitate of the bacterial solution after sonication, lane 4 is the protein sample eluted with 60 mM imidazole, lane 5 is the protein sample eluted with 200 mM imidazole, and lane 6 is the protein sample eluted with 500 mM imidazole;

[0032] Figure 3 It is the SDS-PAGE electrophoresis result diagram of the concentration of the pET22b-TEX264 recombinant plasmid protein. Among them, M is the protein Marker, and lane 1 is the protein band after concentration;

[0033] Figure 4 It is the line graph of the ELISA determination of serum titer;

[0034] Figure 5 It is the SDS-PAGE diagram of the TEX264 antibody, with a purity of 98%;

[0035] Figure 6A and Figure 6B It is the Western blot verification diagram of the polyclonal antibody against rat TEX264 protein. Among them Figure 6A It is the WB verification result diagram of the detection of TEX264 recombinant protein by the TEX264 polyclonal antibody; Figure 6BWB verification result graph for detecting the expression of endogenous TEX264 protein in human carotid intima tissue, rat brain tissue, and mouse brain tissue with TEX264 polyclonal antibody;

[0036] Figure 7 Immunofluorescence verification result graph for detecting the expression of endogenous TEX264 protein in human carotid intima tissue, rat brain tissue, and mouse brain tissue with polyclonal antibody against rat TEX264 protein;

[0037] Figure 8 Immunohistochemical verification result graph for detecting the expression of endogenous TEX264 protein in human carotid intima tissue, rat brain tissue, and mouse brain tissue with polyclonal antibody against rat TEX264 protein. Detailed implementation manners

[0038] The technical solutions in the present invention will be described below in conjunction with the accompanying drawings and embodiments.

[0039] Since there is no commercial anti-rat TEX264 antibody in the prior art, the technical problem to be solved by the present invention is to overcome the current lack of anti-rat TEX264 antibody that can be used for scientific research or applications, and provide a preparation method of anti-rat TEX264 polyclonal antibody. A polyclonal antibody against rat TEX264 is prepared. This antibody can specifically recognize rat endogenous TEX264 protein and is used in experiments such as ELISA, Western Blot, and immunofluorescence analysis. The present invention provides a material basis for studying the function of TEX264.

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

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

[0042] The secondary structure of the full protein sequence (NP_001007666.1) of rat TEX264 in NCBI was analyzed, and the hydrophilicity, hydrophobicity, antigenicity, etc. were analyzed. The 41-220 aa encoded by the 472-1011 bp nucleotide sequence 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 the protein expression level in prokaryotes, the 472-1011 bp nucleotide sequence shown in SEQ ID NO.1 was codon-optimized, and the optimized sequence is shown in SEQ ID NO.3, and the gene fragment was synthesized. The gene fragment synthesis was completed by Tsingke Biotechnology Co., Ltd.

[0044] Among them, the SEQ ID NO.1 sequence is:

[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 engineering bacteria

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

[0052] Subsequently, a transformation experiment was carried out. After thawing the frozen BL21(DE3) competent cells on ice, 0.5 - 1 μg of the recombinant plasmid was added and gently mixed. Then, it was incubated on ice for 30 min. After heat shock in a 42 °C water bath for 90 s, it was immediately placed on ice for 1 - 2 min. Then, 800 μL of pre-warmed LB liquid medium was added, and it was incubated at 37 °C with shaking at 158 rpm for 50 min. After centrifugation (6000 rpm, 4 minutes), 650 μL of the supernatant was removed, and the remaining 150 μL of the bacterial solution was resuspended thoroughly and evenly spread on the LB solid medium containing ampicillin (content: 60 μg / ml). Finally, the plate was inverted and incubated in a 37 °C constant temperature incubator for 12 - 16 h, and positive single colony clones containing the recombinant plasmid were obtained through antibiotic resistance screening.

[0053] Example 3 Expression of Rat TEX264 Recombinant Protein

[0054] 1. Small-scale expression:

[0055] The pET-22b-TEX264 recombinant plasmid vector with correct sequencing was transformed into BL21(DE3) competent cells. A single colony was picked and inoculated into 3 ml of LB liquid medium containing ampicillin (content: 60 μg / ml), and cultured at 37 °C with shaking at 200 rpm. When the OD600 reached 0.6, part of the bacterial solution was transferred to a new tube as the negative control group, and the remaining bacterial solution was added with 1 mM IPTG for induction, and cultured with shaking at 18 °C for 3 h.

[0056] 0.15 mL of the bacterial solution from each of the two groups was taken, centrifuged at 12000×g for 2 min, the bacterial solution precipitate was collected, dispersed with 40 μl of 10 mM Tris-HCl (pH 8.0) solution, and resuspended and lysed with 40 μL of 1×loading buffer, and boiled at 100 °C for 5 min, and then detected by SDS-PAGE electrophoresis to check whether the protein was expressed.

[0057] Experimental results:

[0058] The results of small-scale expression are as Figure 1 shown. It can be seen from Figure 1 that there is a specific expression band at a relative molecular mass of about 32 KDa in the IPTG induction group (lane 3). The experimental results prove that IPTG can indeed induce the expression of the target protein.

[0059] 2. Large-scale expression

[0060] Inoculate 10 μl of the activated bacterial solution from Step 1 into 10 ml of the corresponding resistant LB liquid medium (containing ampicillin antibiotic at a content of 60 μg / ml), and culture it overnight at 37 °C with shaking. Transfer the cultured bacterial solution to 700 ml of the corresponding resistant LB liquid medium, and culture it at 37 °C and 200 rpm until OD600 = 0.6. Add 1 mM IPTG for induction, and continue to culture it with shaking at 18 °C for 4 h. Then centrifuge at 6000 rpm / min for 3 min, collect the bacterial solution precipitate, and resuspend it in 20 mM Tris-HCl, 0.5 M NaCl (pH 8.0) buffer.

[0061] Ultrasonically disrupt the bacterial cells (control the power at 300 W, ultrasonicate for 4 s, pause for 4 s, and ultrasonicate 99 times). Centrifuge at 16000 rpm and 4 °C for 50 min, collect the supernatant and precipitate respectively, resuspend them with 40 μl of 1X SDS-PAGE loading buffer, and after boiling water bath for 5 min, take 10 μl for protein electrophoresis detection, and keep the remaining supernatant and precipitate at 4 °C for standby.

[0062] Experimental results:

[0063] The identification results of the large-scale expression of the pET-22b-TEX264 recombinant plasmid protein are as Figure 2 shown. As can be seen from Figure 2 the figure, there is a specific expression band in the supernatant of the sample pET-22b-TEX264 bacterial cells (lane 2) at a relative molecular mass of approximately 32 KDa, and it has higher specificity than the band in the pET-22b-TEX264 precipitate (lane 3). This indicates that the recombinant plasmid is successfully expressed and mainly exists in the supernatant.

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

[0065] Filter the supernatant solution containing TEX264 protein through a 0.45 μm filter membrane to remove impurities. Equilibrate the Ni-NTA affinity chromatography column (purchased from MCE Company, model hy-k0221) with a buffer containing 20 mM Tris-HCl and 0.5 M NaCl (pH 8.0), and complete the pretreatment of the column material at a flow rate of 1 mL / min. Load the filtered protein sample onto the column at the same flow rate. After the TEX264 protein specifically binds to the nickel ions, continue to wash with the equilibration buffer until the Bradford detection (G250 reagent) shows that there is no protein residue in the effluent (the blue color remains stable). Perform stepwise elution with a gradient imidazole buffer (20 mM, 60 mM, 200 mM, 500 mM), and collect each eluate until the G250 detection solution no longer shows color. After completion of the elution, wash the column material with 3 column volumes of deionized water in sequence, and seal and store the chromatography column with 20% ethanol.

[0066] The elution components at each concentration collected were analyzed by SDS-PAGE electrophoresis to screen for the target protein components with high purity. Finally, they were replaced into PBS buffer (pH 7.4) by dialysis and stored at -80 °C for later use. Among them, the steps of the dialysis method are as follows:

[0067] (1) Pretreatment of the dialysis bag (Fusheng A1999764): The new dialysis bag was treated with 10% glycerol to remove sulfides, heavy metals, and some impurities with ultraviolet absorption. The dialysis bag was cut into small segments of 100 - 120 mm, boiled in 50% ethanol for 1 h, then washed successively with 50% ethanol, 0.01 mol / L sodium bicarbonate, and 0.001 mol / L EDTA solution, and finally rinsed with distilled water 3 - 5 times. One end of the dialysis bag was clamped with a dialysis bag clip, filled with water from the other end, and slightly pressed with fingers to check for leakage. The treated dialysis bag was stored in distilled water for later use.

[0068] (2) Subsequently, 5 mL of the supernatant solution containing TEX264 protein was taken, and an equal volume of saturated ammonium sulfate was added and allowed to stand for 15 minutes to precipitate the protein. The reversibility of the precipitate was verified through a redissolution experiment. Finally, the protein solution containing salt was injected into the dialysis bag and suspended in distilled water, and the Cl - (silver nitrate method) and protein leakage (biuret reaction) were detected at intervals. The water was changed every 20 minutes until the Cl - was completely removed. After dialysis was completed, the protein precipitation phenomenon inside the bag was recorded.

[0069] Experimental results:

[0070] The protein purification effect was identified by SDS-PAGE electrophoresis as Figure 2 shown. Among them, lane 4 was the protein sample eluted with 60 mM imidazole, lane 5 was the protein sample eluted with 200 mM imidazole, and lane 6 was the protein sample eluted with 500 mM imidazole. From Figure 2 lanes 4 to 6, it can be seen that the protein band eluted with 200 mM imidazole was relatively single.

[0071] The results of the specific bands after protein concentration can be seen in Figure 3 . From Figure 3 it can be seen that a single bright band was obtained after concentration and purification, indicating that a recombinant protein with high purity was obtained after purification.

[0072] Example 5 Preparation of Polyclonal Antibody Against Rat TEX264 Protein

[0073] 1. Immunization of New Zealand White Rabbits

[0074] Primary immunization: New Zealand white rabbits weighing about 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 normal saline, and then an equal volume of Freund's adjuvant was added and mixed evenly to form an oil emulsion by emulsification. The rabbits were immunized by subcutaneous injection on the back at 8 points.

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

[0076] The immunization cycle was 49 days, and the rabbits were immunized 4 times in total. For the first immunization, Freund's complete adjuvant (purchased from MCE, product number HY-153808) was used for emulsification. For the second, third, and fourth immunizations at the 2nd, 4th, and 6th weeks, Freund's incomplete adjuvant (purchased from MCE, product number HY-153808A) was used for emulsification. The immunization dose was 500 μg. Blood was collected for detection one week after the 3rd immunization, and a large amount of blood was collected one week after the 4th immunization. The collected blood was inactivated at 37 °C for 2 h, then left to clot overnight at 4 °C to release serum. After centrifugation at 5000 rpm / min for 10 min, the supernatant was collected after two centrifugations, which was the serum.

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

[0078] The TEX264 protein was diluted to 2 μg / ml. Using carbonate buffer as the coating solution, 100 μl of the coated protein was added to a 96-well plate and incubated overnight at 4 °C. The next day, each well was washed 3 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 blocked at 37 °C for 1 hour, and washed 3 times again with PBST buffer.

[0079] Negative control (using blank serum as the negative control) and post-immune serum were 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, with three replicates for each 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 (i.e., the TEX264 polyclonal antibody of the present invention) bound to the antigen, and incubate at 37 °C for 40 minutes. After washing three times with PBST buffer, wash twice with ultrapure water, then add 100 µl of TMB chromogenic solution (Beyotime, P0209), incubate for 20 minutes in a dark room temperature environment, and then add 100 µl of hydrochloric acid to terminate the reaction. Finally, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance (A450) value of each well at 450 nm. Determine the highest dilution factor of the diluted antiserum when the absorbance at 450 nm is more than 2.1 times that of the pre-immune serum by reading, that is, the titer = the highest dilution factor when OD antiserum / OD pre-immune serum ≥ 2.1.

[0081] Experimental results:

[0082] The detection results of the titer are as Figure 4 shown. It can be seen from Figure 4 this that the titer of the antiserum (polyclonal antibody) is about 128K. This shows that the polyclonal antibody against rat TEX264 protein has been successfully prepared and has a high titer.

[0083] 3. Antibody purification and purity detection: Dialyze 10 mg of TEX264 protein against 20 mM sodium bicarbonate buffer at 4 °C for 16 h, centrifuge at 10000 rpm for 10 min at 4 °C, take the supernatant, and couple it with cyanogen bromide-activated column material (Dalian Jingjian Biotechnology Co., Ltd., 09250) overnight at 4 °C. Wash the column bed successively with 20 mM sodium bicarbonate and Tris buffer, and store it in NaCl-sodium azide buffer (TSA buffer (PH = 8), REGETE B0091) after blocking. Verify the coupling efficiency by SDS-PAGE.

[0084] After equilibration of the antigen column, take the antiserum sample prepared in Step 1, centrifuge at 12,000 rpm for 10 min at 4 °C, collect the supernatant, and filter it through a 0.45 µm filter membrane. After mixing the pretreated antiserum sample with sodium acetate buffer in a ratio of 1:4, load the sample at a rate of 0.5 mL / min. Wash the column with sodium acetate buffer (0.2 mol / L sodium acetate and 0.0002 mol / L sodium hydroxide) until the detection with G250 shows no color. Wash the column bed with ice acetic acid elution buffer (0.1 mol / L ice acetic acid and 0.1 mol / L sodium acetate), collect the elution peak, and quickly adjust the pH of the elution peak to neutral with saturated sodium carbonate. Concentrate the eluate by ultrafiltration to the original volume, centrifuge at 12,000 rpm for 10 min at 4 °C, collect the supernatant, store it temporarily at 4 °C, and perform SDS-PAGE detection. Analyze the purity by SDS-PAGE Coomassie Brilliant Blue staining and Bandscan software, and finally aliquot and store the antibody at -20 °C.

[0085] Experimental results:

[0086] The results of the specific bands of the purified polyclonal antibody can be seen in Figure 5 . From Figure 5 it can be seen that 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 Polyclonal Antibody Against Rat TEX264 Protein for Recognizing TEX264 Protein

[0088] 1. Sample preparation

[0089] 1) Protein samples: Take 5, 10, 20, 40 ng of rat TEX264 recombinant protein samples; take 0.1 g of human carotid intima tissue, rat brain tissue, and mouse brain tissue, add 1 mL of protein lysate (Solarbio, R0020), homogenize with a tissue grinder, lyse at 4 °C for 30 min, centrifuge at 12,000 rpm for 10 min, take the supernatant, and store it at -80 °C after BCA protein quantification.

[0090] 2) Paraffin sections: Fix human carotid intima tissue, rat brain tissue, and mouse brain tissue in 4% paraformaldehyde for 4 h, dehydrate them successively by soaking in gradient alcohol (50% → 70% → 80% → 90% → 95% → 100%), clarify them by soaking in xylene for 30 min successively, embed them in a mold after impregnation with paraffin to form a wax block, and cut 5-μm-thick sections for storage.

[0091] 2. Specificity detection

[0092] 1) Immunoblotting:

[0093] The protein sample was separated by 12% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. The transfer conditions were a constant current of 60 mA for 80 min. After membrane transfer, it was immediately blocked with PBST buffer at room temperature for 2 h.

[0094] The membrane was immersed in the primary antibody working solution prepared from the polyclonal antibody against rat TEX264 protein obtained in Example 5 (the antibody was diluted 1:1000 in PBST) and incubated at room temperature for 1 h, then washed 4 times with PBST, 10 min each time. HRP-labeled secondary antibody (the HRP-labeled secondary antibody was purchased from Affinity, model S0001) was added and incubated at room temperature for 1 h, and then washed again 4 times with PBST, 10 min each time. ECL chemiluminescence method was used for development.

[0095] Experimental results:

[0096] The experimental results of the TEX264 recombinant protein sample of the present invention are as Figure 6A shown. As can be seen from Figure 6A , when the positive serum of immunized big rabbits was used as the primary antibody, a specific single band of TEX264 recombinant protein appeared at 32 kDa. The WB observation value was consistent with the SDS-PAGE observation value, and the quality control passed. The TEX264 in human carotid artery intima, rat brain tissue and mouse brain tissue is as Figure 6B shown. As can be seen from Figure 6B , when the positive serum of immunized big rabbits was used as the primary antibody, an obvious specific band was detected at 32 kDa in human carotid artery intima, rat brain tissue and mouse brain tissue. This shows that the polyclonal antibody against rat TEX264 protein provided by the present invention can not only specifically recognize rat TEX264 protein, but also specifically recognize human and mouse TEX264 proteins. This polyclonal antibody has high specificity and multi-species reactivity.

[0097] 2) Immunofluorescence:

[0098] First, dewax the paraffin sections through a xylene gradient and hydrate them through an ethanol gradient to distilled water. Subsequently, perform antigen retrieval using citrate (pH 6.0) or EDTA (pH 9.0) buffer, heating in stages at medium-high power in a microwave (for a total of 15 minutes) or treating with a pressure cooker (3 minutes of steam). After retrieval, allow to cool naturally and wash 3 times with PBS. Incubate the sections with Triton X-100 for 30 minutes for permeabilization, then block with 1% BSA for 30 minutes. Then add the diluted polyclonal antibody against rat TEX264 protein (dilution factor is 1:300) and incubate overnight at 4°C in a humidified chamber. The next day, wash with PBS, add the corresponding FITC-labeled goat anti-rabbit secondary antibody (Proteintech, SA00003-2) and incubate in the dark at room temperature for 50 minutes. After staining with DAPI for 10 minutes to label the cell nuclei, treat with a spontaneous fluorescence quencher (Beijing Pulilai Gene Technology Co., Ltd., C1212) for 5 minutes to eliminate background interference. Finally, wash thoroughly with PBS, mount with an anti-fluorescence quenching mounting medium (Beijing Solarbio Science & Technology Co., Ltd., S2100), and store in the dark at 4°C. When interpreting the results, DAPI shows blue cell nuclei, and the positive signal appears green according to the fluorescence labeling.

[0099] Experimental results:

[0100] The experimental results are as Figure 7 shown. It can be seen from Figure 7 that when using the positive serum of immunized rabbits as the primary antibody, green-labeled TEX264 protein signals and blue-labeled cell nucleus signals appear in human carotid artery intima, rat brain tissue, and mouse brain tissue. This indicates that the polyclonal antibody against rat TEX264 protein provided by the present invention can be used for immunofluorescence detection and has multi-species reactivity.

[0101] 3) Immunohistochemistry:

[0102] First, dewax the paraffin sections through a xylene gradient and hydrate them through an ethanol gradient to distilled water. Subsequently, perform antigen retrieval using citric acid (pH 6.0) or EDTA (pH 9.0) buffer, heating in stages at medium-high power in a microwave (15 minutes in total) or treating with a pressure cooker (3 minutes of steam). After retrieval, cool naturally and wash 3 times with PBS. To eliminate background interference, block endogenous peroxidase in the sections with 3% hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd., 10011218) for 25 minutes. After washing with PBS, block at room temperature with goat serum (Beyotime Biotechnology, C0265) for 15 minutes. Add the diluted primary antibody (polyclonal antibody against rat TEX264 protein, dilution factor 1:300) and incubate overnight at 4°C in a wet box. The next day, after washing with PBS, add the HRP-labeled secondary antibody (ZSGB-BIO, ZB-2306) and incubate at room temperature for 50 minutes. Control the color development time of the DAB chromogenic solution (ZSGB-BIO, ZLI-9018) under the microscope (brown-yellow is positive), and terminate the reaction with tap water. Counterstain the cell nuclei with hematoxylin (Zhuhai Biosino Biotechnology Co., Ltd., BA4041) for 5 - 10 minutes. After differentiation with hydrochloric acid alcohol and bluing with ammonia water, dehydrate and clear the sections through a gradient of alcohol and xylene, and mount with neutral balsam. Final interpretation: The cell nuclei are blue (hematoxylin staining), and the positive signal is brown-yellow (DAB chromogenic reaction).

[0103] Experimental results:

[0104] The experimental results are as Figure 8 shown. As can be seen from Figure 8 this, when using the positive serum of immunized rabbits as the primary antibody, brown-labeled TEX264 protein signals and blue-labeled cell nucleus signals appear in human carotid artery intima, rat brain tissue, and mouse brain tissue. This shows that the polyclonal antibody against rat TEX264 protein provided by the present invention can be used for immunohistochemical detection and has reactivity with multiple species.

[0105] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A recombinant protein of rat TEX264, characterized in that The amino acid sequence of the rat TEX264 recombinant protein is shown in SEQ ID NO.

2.

2. A recombinant vector expressing the recombinant protein of rat TEX264 according to claim 1, characterized in that The recombinant vector contains a DNA sequence encoding the rat TEX264 recombinant protein, and the DNA sequence is shown in SEQ ID NO.

3.

3. The recombinant vector according to claim 2, characterized in that The plasmid used for constructing the recombinant vector is the pET-22b expression vector.

4. A method for preparing the recombinant protein of rat TEX264 according to claim 1, characterized in that The method includes: 1) Transforming the recombinant vector according to any one of claims 2-3 into competent cells to construct an engineered bacterium carrying the recombinant vector; 2) Inducing the engineered bacterium with IPTG to express the rat TEX264 recombinant protein and purifying it.

5. A polyclonal antibody against rat TEX264 protein, characterized in that The polyclonal antibody is prepared by immunizing an animal with the rat TEX264 recombinant protein according to claim 1.

6. A method for preparing the polyclonal antibody against rat TEX264 protein according to claim 5, characterized in that It includes: (1) Preparing the rat TEX264 recombinant protein according to claim 1; (2) Using the rat TEX264 recombinant protein obtained in step (1) as an antigen to immunize an animal; (3) After immunizing the animal in step (2), obtaining animal serum and purifying it to obtain the polyclonal antibody against the rat TEX264 protein.

7. The preparation method according to claim 6, characterized in that The animal in step (2) is a rabbit.

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

9. The application of the polyclonal antibody against rat TEX264 protein according to claim 5 in a specific detection method of rat TEX264 protein, wherein the specific detection method refers to: a detection method using the polyclonal antibody against rat TEX264 protein to recognize rat TEX264 protein.

10. A specific detection method of TEX264 protein, characterized in that It includes: (1) Obtaining a protein sample: The protein sample is the TEX264 protein; (2) Transferring the protein sample to a PVDF membrane after SDS-PAGE gel electrophoresis and blocking it with PBST buffer at room temperature; (3) Diluting the polyclonal antibody against the rat TEX264 protein according to claim 5 in PBST buffer to prepare a working solution of the primary antibody, immersing the PVDF membrane in step (2) in the working solution of the primary antibody, incubating at room temperature, and washing; (4) Adding an HRP-labeled secondary antibody, incubating at room temperature, and washing; (5) Exposing with a luminescent solution and observing the results.

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