Polyclonal Antibody of Porcine TMEM106a Protein, Its Preparation Method and Application

Abstract

This invention discloses a polyclonal antibody against porcine TMEM106a protein, its preparation method, and its applications, belonging to the field of biotechnology. The porcine TMEM106a protein polyclonal antibody is prepared by immunizing animals with the protein sequence shown in SEQ ID NO.2 as the antigen. This invention utilizes a truncated segment of the porcine TMEM106a protein to prepare a porcine TMEM106a protein polyclonal antibody. The porcine TMEM106a protein polyclonal antibody prepared by this invention exhibits high specificity and titer, and can specifically recognize the TMEM106a protein in porcine cells, thus enabling its application in porcine TMEM106a protein detection reagents or kits. This provides important tools and technical support for functional studies of the TMEM106a protein in pigs and is suitable for large-scale production and application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a polyclonal antibody against porcine TMEM106a protein, its preparation method, and its application. Background Technology

[0002] Transmembrane protein 106a is a primary transmembrane protein located on the plasma membrane and in mitochondria. It participates in physiological processes including glycoprotein biosynthesis, positive regulation of cytokine production, and positive regulation of intracellular signal transduction. It also participates in macrophage polarization through the MAPK and NF-κB signaling pathways. Due to its role in inhibiting cell proliferation and reducing migration in certain cancers, it has been identified as a tumor suppressor and is an important regulatory protein for cellular life activities.

[0003] Currently, commercially available antibodies against TMEM106a protein are mainly targeted at humans or rabbits. These antibodies have a weak ability to specifically recognize TMEM106a protein in porcine tissues, which greatly limits the research and application of TMEM106a in pigs. Therefore, developing an antibody that can specifically recognize TMEM106a protein in porcine cells is of great research significance. Summary of the Invention

[0004] The purpose of this invention is to provide a polyclonal antibody against porcine TMEM106a protein, its preparation method, and its applications, thereby addressing the problems existing in the prior art. This invention utilizes a truncated segment of porcine TMEM106a protein to prepare a polyclonal antibody against porcine TMEM106a protein. The porcine TMEM106a protein polyclonal antibody prepared by this invention exhibits high specificity and titer, and can specifically recognize TMEM106a protein in porcine cells, thus enabling its application in porcine TMEM106a protein detection reagents or kits. This provides important tools and technical support for functional studies of TMEM106a protein in pigs and is suitable for large-scale production and application.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a porcine TMEM106a protein polyclonal antibody, which is prepared by immunizing animals with the protein of the sequence shown in SEQ ID NO.2 as the antigen.

[0007] The present invention also provides a method for preparing the above-mentioned porcine TMEM106a protein polyclonal antibody, comprising the following steps:

[0008] (1) A recombinant expression vector was prepared by using the coding gene of the truncated intramembrane region of porcine TMEM106a. The recombinant expression vector was transferred into host cells, induced to express, purified, and porcine TMEM106a recombinant protein was obtained.

[0009] (2) Using the porcine TMEM106a recombinant protein as an antigen, immunize the animals, collect the serum, and obtain the porcine TMEM106a protein polyclonal antibody.

[0010] Furthermore, the nucleotide sequence of the gene encoding the truncated intramembrane region of the porcine TMEM106a is shown in SEQ ID NO.1.

[0011] Optionally, the recombinant expression vector is a prokaryotic recombinant expression vector.

[0012] Preferably, the starting vector of the prokaryotic recombinant expression vector is the pET-28a(+) vector.

[0013] Optionally, the host cell is a BL21(DE3) cell.

[0014] Furthermore, the method for inducing expression is the IPTG method.

[0015] Optionally, the final concentration induced by IPTG is 0.2 mM.

[0016] Optionally, the induction conditions are 5-20 h at 16-37℃.

[0017] Preferably, the induction conditions are 5 hours at 37°C.

[0018] Optionally, the purification method includes Ni 2+ Affinity chromatography column separation and purification.

[0019] Optionally, the animal is a mouse.

[0020] The present invention also provides the application of the above-mentioned porcine TMEM106a protein polyclonal antibody or the porcine TMEM106a protein polyclonal antibody prepared by the above preparation method in the preparation of products for detecting TMEM106a protein.

[0021] Furthermore, the product includes a kit for detecting the expression of TMEM106a protein in porcine cells or tissues.

[0022] Optionally, the kit includes a Western Blot kit.

[0023] The present invention discloses the following technical effects:

[0024] This invention designs and screens suitable target nucleotides, extracts a truncated segment of porcine TMEM106a protein, and prepares a porcine TMEM106a protein polyclonal antibody using gene recombination technology. Experiments show that the porcine TMEM106a protein polyclonal antibody prepared in this invention has high specificity, capable of specifically recognizing TMEM106a protein in porcine cells, thus making it applicable in porcine TMEM106a protein detection reagents or kits. The raw materials used in this gene recombination are inexpensive, readily available, and produce good animal immunization effects and high antibody titers, effectively ensuring the quality of commercialized porcine TMEM106a protein polyclonal antibodies. This provides important tools and technical support for functional studies of TMEM106a protein in pigs and is suitable for large-scale production and application. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the 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.

[0026] Figure 1 The image shows the predicted results for the intramembrane, transmembrane, and extramembrane regions of the TMEM106a protein.

[0027] Figure 2 Figure 1 shows the results of His-TMEM106a-in fusion protein expression identification. Lane M represents the protein marker; lane 1 represents uninduced host cell BL21 (pET-28a(+)-TMEM106a); lane 2 represents whole-cell sample of host cell BL21 (pET-28a(+)-TMEM106a) induced at 16℃ for 20 h; lane 3 represents the supernatant after cell lysis in lane 2; lane 4 represents the precipitate after cell lysis in lane 2; lane 5 represents the whole-cell sample of host cell BL21 (pET-28a(+)-TMEM106a) induced at 37℃ for 5 h; lane 6 represents the supernatant after cell lysis in lane 5; and lane 7 represents the precipitate after cell lysis in lane 5. The arrows indicate the target protein.

[0028] Figure 3 This image shows the purification results of the His-TMEM106a-in fusion protein. In the image, A shows the purified protein sample after induction at 37℃ for 5 h, and B shows the purified protein sample after induction at 16℃ for 20 h. Lane M is the protein marker, lane 1 is the supernatant after disruption, lane 2 is the precipitate after disruption, lane 3 is the resuspended gel, lane 4 is the flow-through buffer, lane 5 is the washing buffer, and lanes 6, 7, and 8 are the purified His-TMEM106a-in fusion protein collected after elution. The arrows indicate the target protein.

[0029] Figure 4 The image shows the Western Blot results of the purified His-TMEM106a-in fusion protein. In the image, A shows the results of the identification using His-tagged antibody as the primary antibody, and B shows the results of the identification using the self-made TMEM106a antibody as the primary antibody. Lane M is the protein marker, and lanes 1 and 2 are the purified His-TMEM106a-in fusion protein. The arrows indicate the target protein.

[0030] Figure 5 Figure showing the results of detecting TMEM106a protein in porcine alveolar macrophages (PAM) and porcine macrophages (3D4 / 21) using a polyclonal antibody against porcine TMEM106a protein.

[0031] Figure 6 Immunofluorescence sections of porcine TMEM106a polyclonal antibody and commercially available rabbit TMEM106a polyclonal antibody are shown; the scale bar is 100 μm. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0038] Example 1

[0039] This embodiment provides a porcine TMEM106a protein polyclonal antibody to promote the research and application of TMEM106a in pigs. The preparation of the porcine TMEM106a protein polyclonal antibody mainly includes three processes: cloning the porcine TMEM106a gene, expression and purification of the recombinant porcine TMEM106a protein, and preparation of the porcine TMEM106a protein polyclonal antibody. The specific operation process is as follows:

[0040] I. Cloning of the porcine TMEM106a gene

[0041] The gene CDS sequence was obtained from NCBI, and the intramembrane, transmembrane, and extramembrane regions of the TMEM106a protein were identified using the website developed by the Technical University of Denmark (https: / / dtu.biolib.com / DeepTMHMM). Figure 1 As shown. According to Figure 1 As shown in the results, since the transmembrane chemical structure of transmembrane proteins is similar to that of signal peptides, and the organelles contained in prokaryotic cells are relatively simple, it is difficult for them to complete a series of complex processes such as recognition, excision, guidance of endoplasmic reticulum and Golgi repackaging and secretion of signal peptides as in eukaryotic cells. Therefore, the intramembrane region gene of TMEM106a was specially selected and optimized using Escherichiacoli. The sequence shown in SEQ ID NO.1 was synthesized by a biotechnology company, and the sequence shown in SEQ ID NO.1 was cloned into the vector pET-28a(+) to construct a recombinant expression plasmid, which was named: pET-28a(+)-TMEM106a.

[0042] SEQ ID NO.1:

[0043] atgggcgagacattctcccagctgggctctcagaaggatgagaacaagtccatcttgccccctgacccaaccatcggcaggaagtcggccagctactccagcaccaacagcagcaaggttttttgttcctgtgtgcct tgtgaaagggccactggtgccagctttgttatttgtcccacatgccagggcagtggggagatccctcgagagctagagaagcagctggtggccctcatcccctatggggaccagaggctgaagcccaggcacacgaag.

[0044] II. Expression and purification of porcine TMEM106a recombinant protein

[0045] 10 μl of the recombinant expression plasmid pET-28a(+)-TMEM106a was added to 100 μl of BL21(DE3) competent cells (host cells). After being placed on ice for 30 min, the cells were heat-shocked at 42℃ for 1 min, then quickly placed on ice for 2 min. 0.9 ml of LB medium was added, and the cells were shaken at 37℃ and 180 r / min for 45 min. The cells were then plated on NA plates containing 50 μg / ml kanamycin. Positive clones were selected for enzyme digestion and identification. The obtained host cells were named BL21(pET-28a(+)-TMEM106a).

[0046] The expression of His-TMEM106a-in fusion protein (recombinant protein antigen) in host cells was induced using the IPTG induction method, as follows:

[0047] Host cells BL21 (pET-28a(+)-TMEM106a) were picked and inoculated into 1 ml LB medium containing 50 μg / ml kanamycin, and incubated overnight at 37°C and 180 r / min with shaking. The next day, the cells were inoculated into 25 ml LB medium containing 50 μg / ml kanamycin at a 1:100 ratio, and incubated for 2 hours at 37°C and 180 r / min with shaking until the cell OD reached 100. 600 It is 0.6-0.8.

[0048] Take 1 ml of culture and centrifuge at 10000 rpm for 2 min at room temperature. Discard the supernatant and resuspend the bacterial pellet in 80 μl PBS and 20 μl 5×SDS PAGE loading buffer. Then, add IPTG to the remaining culture to a final concentration of 0.2 mM and shake at 16°C and 180 rpm for 20 h, or at 37°C and 180 rpm for 5 h, to induce fusion protein expression.

[0049] Then, take 1 ml of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, resuspend the bacterial pellet in 100 μl of 1×SDS loading buffer, centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the pellet in PBS.

[0050] After ultrasonic disruption of the resuspension, the supernatant and precipitate were resuspended in loading buffer, and then analyzed by 12% SDS-PAGE with Coomassie brilliant blue staining. The results are as follows: Figure 2 As shown, the target proteins induced at 16 ℃ and 37 ℃ were mainly found in the bacterial cell precipitate.

[0051] Using Ni 2+ The His-TMEM106a-in fusion protein was separated and purified using affinity chromatography, and the specific procedures are as follows:

[0052] First, take 8 ml of a well-mixed 50% BeyoGold solution. TM His-tag Purification Resin was centrifuged at 4°C, the stock solution was discarded, and the sample was resuspended in denaturing lysis buffer. After centrifugation at 1000g for 10s, 20 μl was collected. 30 ml of bacterial lysis supernatant was added to the resuspended sample, and the mixture was incubated overnight at 4°C on a side-shaking shaker with gentle shaking. The lysis buffer and BeyoGold were then added... TM The His-tag Purification Resin mixture was loaded into an empty affinity chromatography column tube. The column cap was opened, and the liquid was allowed to flow out under gravity, collecting 20 μl of the flow-through. The column was washed with 8 ml of denaturing wash buffer, collecting 20 μl of the wash buffer. The target protein was eluted three times with 4 ml of elution buffer, collecting 20 μl of the elution buffer each time. Finally, 12% SDS-PAGE analysis was performed, and the results are shown below. Figure 3 As shown, the purified His-TMEM106a-in fusion protein has high purity.

[0053] Western blot analysis was performed on the purified His-TMEM106a-in fusion protein using His-tagged antibody as the primary antibody and HRP-labeled goat anti-mouse IgG polyclonal antibody (purchased from TransGen Biotech Ltd.) as the secondary antibody. After elution with protein eluent, the protein was incubated again with a self-made TMEM106a antibody (obtained by injecting mice with a mixture of prokaryotically expressed and purified His-TMEM106a-in fusion protein and adjuvant in equal proportions, followed by three immunizations and extraction of blood supernatant) as the primary antibody and HRP-labeled goat anti-mouse IgG polyclonal antibody as the secondary antibody. Results are shown in the figure. Figure 4 As can be seen, the target band appeared at the expected size.

[0054] The amino acid sequence of the His-TMEM106a-in fusion protein prepared in this invention, as shown in SEQ ID NO.2, was obtained by sequencing.

[0055] SEQ ID NO.2:

[0056] MGETFSQLGSQKDENKSILPPDPTIGRKSASYSSTNSSKVFCSCVPCERATGASFVICPTCQGSGEIPRELEKQLVALIPYGDQRLKPRHTK.

[0057] III. Preparation of Polyclonal Antibody against Porcine TMEM106a Protein

[0058] 1. Animal immunization

[0059] After the concentration of the purified His-TMEM106a-in fusion protein was determined using a BCA concentration assay kit, three C57BL / 6 mice (20-25g) were immunized subcutaneously with 150μg per immunization. For the first immunization, the antigen was mixed with an equal volume of complete Freund's adjuvant. For the second and third immunizations, the antigen was mixed with an equal volume of incomplete Freund's adjuvant. A total of three immunizations were performed, with an interval of two weeks between each immunization.

[0060] 2. Preparation of antiserum

[0061] After immunization, blood was collected for testing. The titer of the antiserum against the purified TMEM106a protein was determined by indirect ELISA. When the titer was greater than 1:50000 (around 7 days after the last immunization), the final blood was collected to prepare the antiserum, which is the porcine TMEM106a protein polyclonal antibody.

[0062] The prepared porcine TMEM106a protein polyclonal antibody was incubated overnight at 4°C in PBS, and its concentration and titer were determined the following day. The specific procedures are as follows:

[0063] (1) Concentration determination: The concentration of the obtained TMEM106a antibody was determined using a BCA concentration assay kit, and the result was 0.8 mg / mL. The purity of the purified antibody was observed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The results showed that the purity of the purified TMEM106a antibody was above 85%.

[0064] (2) Potency determination: The coating solution was prepared by adding 1.59 g of Na2CO3 and 1.93 g of NaHCO3 to 1 L of ultrapure water. The purified His-TMEM106a-in protein antigen was diluted to 4 μg / ml with the coating solution, and 100 μl was added to each well. The plate was refrigerated overnight at 4℃. The coating solution was discarded the next day. The plate was washed 3 times with TBST for 5 min each time. Then, 200 μl of 5% skim milk powder blocking solution was added to each well and the plate was blocked at 37℃ for 1 h. The plate was removed and the inner solution was discarded. The plate was washed 5 times with TBST for 3 min each time. The antibody to be tested was diluted 1:200, then diluted to 1:4000, and then serially diluted to 1:512,000, with 100 μl added to each well. A negative control well containing untreated mouse serum was included. The plate was incubated at 37℃ for 2 h. The plate was removed and the inner solution was discarded. The plate was washed 5 times. Add 100 μl of diluted HRP-labeled goat anti-mouse IgG (1:50000 dilution) enzyme-labeled secondary antibody to each well, incubate at 37°C for 1 h, then remove the plate, discard the internal liquid, and wash the plate 5 times with TBST; add 100 μl of TMB chromogenic solution to each well, incubate at 37°C for 15 min; add 100 μl of TMB chromogenic stop solution to each well to stop the reaction, and immediately read the 450 nm value on the microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0065] The titer test results are shown in Table 1. It can be seen that the purified TMEM106a antibody has a titer greater than 512000, which is appropriate.

[0066] Table 1. Results of indirect ELISA detection of TMEM106a antibody

[0067]

[0068] Example 2

[0069] This embodiment uses the porcine TMEM106a protein polyclonal antibody prepared in Example 1 to detect the expression of TMEM106a protein in porcine cells and tissues, in order to verify the application effect of the porcine TMEM106a protein polyclonal antibody. The specific operation is as follows:

[0070] I. Detection of TMEM106a protein in porcine cells

[0071] 1. Normally cultured porcine alveolar macrophages (PAM) and porcine macrophages (3D4 / 21) were collected and cultured in 6-well plates. 200 μl of RIPA lysis buffer (purchased from Beyotime) and a protease inhibitor at a final concentration of 1 mmol / L were added to each well. After reacting at 4℃ for 15 min, the liquid was transferred to a 1.5 ml centrifuge tube and centrifuged at 12000 r / min and 4℃ for 5 min. The supernatant was collected, and the protein concentration was determined using a BCA concentration kit.

[0072] 2. After preparing 12.5% ​​stacking gel and 12.5% ​​separating gel, load 30 μg of protein into each well; maintain a constant voltage of 90V for the stacking gel and 120V for the separating gel. When the bromophenol blue reaches the bottom of the gel, electrotransfer the sample onto a PVDF membrane (21V constant voltage, 40 min); after electrotransfer, place the PVDF membrane in 5% skim milk blocking buffer and block at 37℃ for 2 h; then dilute the purified porcine TMEM106a protein polyclonal antibody to 1:1000 with the primary and secondary antibody dilution buffer, and rabbit anti-Na... + / K + - Dilute the ATPase internal control primary antibody polyclonal antibody (abmart) to 1:1000 and incubate overnight at 4°C.

[0073] 3. Wash the membrane three times with TBST, 5 min each time; dilute the HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies to 1:1000 with the primary and secondary antibody dilution buffer, and incubate at 4℃ for 2 h; wash four times with TBST, 10 min each time; develop color with Tanon ECL chemiluminescence solution and fix; scan the PVDF membrane and analyze the gray values ​​of the bands using ImageJ software. The results are shown in [Figure number missing]. Figure 5 And Table 2.

[0074] Depend on Figure 5 As can be seen, the porcine TMEM106a protein polyclonal antibody prepared in this invention can specifically detect TMEM106a protein at the expected size.

[0075] As shown in Table 2, the porcine TMEM106a protein polyclonal antibody prepared in this invention can detect the expression of TMEM106a protein in different types of porcine macrophages.

[0076] Table 2. Results of Western Blot analysis of TMEM106a protein expression in porcine cells.

[0077]

[0078] II. Detection of TMEM106a protein in porcine tissue

[0079] Immunofluorescence was performed on healthy pig spleen tissue sections to compare the immunofluorescence effects of the porcine TMEM106a protein polyclonal antibody prepared in this invention with those of a commercially available rabbit polyclonal antibody (purchased from Xinjin Biotechnology Co., Ltd.). The specific procedures are as follows:

[0080] 1. Dewaxing paraffin sections to water: Immerse the sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash in distilled water.

[0081] 2. Antigen retrieval: Retrieval conditions include heat retrieval in a water bath for 40 min. During retrieval, excessive evaporation of the buffer solution should be prevented; the slides must not be allowed to dry. After retrieval, allow the slides to cool naturally. Place the slides in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 min each time.

[0082] 3. Circle blocking with serum: After slightly drying the slide, draw a circle around the tissue with a histochemical pen, add BSA (use 10% donkey serum for primary antibody from goat, and 3% BSA for primary antibody from other sources), and block for 30 min.

[0083] 4. Add primary antibody: Add primary antibody diluted 1:100, place the slice flat in a humidified chamber and incubate overnight at 4°C.

[0084] 5. Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 min each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 min.

[0085] 6. Counterstaining cell nuclei with DAPI: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker for 5 minutes each time. Add DAPI staining solution and incubate at room temperature in the dark for 10 minutes.

[0086] 7. Quenching of tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 min each time. Add autofluorescence quencher solution B for 5 min, and rinse with running water for 10 min.

[0087] 8. Mounting: Mount the slide with anti-fluorescence quenching mounting medium.

[0088] 9. Image acquisition: DAPI excitation wavelength 330-380 nm, emission wavelength 420 nm; 488 excitation wavelength 465-495 nm, emission wavelength 515-555 nm; CY3 excitation wavelength 510-560 nm, emission wavelength 590 nm; CY5 excitation wavelength 608-648 nm, emission wavelength 672-712 nm.

[0089] See results Figure 6 As can be seen, both the porcine TMEM106a protein polyclonal antibody (self-made TMEM106a) prepared in this invention and the commercial antibody (commercial TMEM106a) can specifically recognize the TMEM106a protein in the same porcine spleen tissue, and the fluorescence intensity of the antibody prepared in this invention is significantly improved compared with the commercial antibody.

[0090] In summary, this invention has prepared a polyclonal antibody against porcine TMEM106a protein, which can specifically recognize TMEM106a protein in different types of porcine cells, providing a guarantee and technical support for the in-depth research and application of porcine TMEM106a.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a porcine TMEM106a protein polyclonal antibody in the preparation of products for detecting TMEM106a protein, characterized in that, The porcine TMEM106a protein polyclonal antibody was prepared by immunizing an animal with the protein sequence shown in SEQ ID NO.2 as the antigen; the animal was a mouse. The preparation method of the antigen is as follows: A recombinant expression vector was prepared using the coding gene of porcine TMEM106a with a truncated intramembrane region. This recombinant expression vector was then transformed into host cells, induced to express, and purified to obtain porcine TMEM106a recombinant protein, which serves as the antigen. The purification method includes Ni... 2+ Affinity chromatography column separation and purification; The nucleotide sequence of the gene encoding the truncated intramembrane region of the porcine TMEM106a is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The method for inducing expression is the IPTG method.

3. The application according to claim 1, characterized in that, The conditions for inducing expression are: induction at 16-37℃ for 5-20 hours.

4. The application according to claim 1, characterized in that, The product includes a kit for detecting the expression of TMEM106a protein in porcine cells or tissues.

5. The application according to claim 4, characterized in that, The kit includes a Western Blot kit.

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