Antibody for resisting Blackett black cattle MSTRG.25151 protein

Abstract

The invention belongs to the technical field of antibody preparation, and provides an antibody for resisting a Blackett black cattle MSTRG.25151 protein. The antibody provided by the invention has the advantages of high specificity, clear WB band and no cross reaction, and provides a tool for functional research and protein expression detection of the MSTRG.25151 gene.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibody preparation, and particularly relates to a recombinant monoclonal antibody of Brucella melitensis MSTRG.25151 protein. BACKGROUND

[0002] The information disclosed in this background section is intended to increase an understanding of the general context of the application and is not necessarily recognized as prior art by the applicant.

[0003] In modern beef cattle breeding industry, reproductive performance as one of the core economic traits that determines the economic benefits of breeding directly affects the propagation efficiency, production cycle and breeding income of beef cattle population, and plays a crucial role in promoting the development of beef cattle industry in a large scale and intensive manner. For a breeding bull, the level of its reproductive performance is mainly determined by the quality of semen, and the normal development of testis and the process of spermatogenesis in testis tissue is the premise to ensure the quality of semen. If the testis development is abnormal, such as insufficient testis weight and incomplete development of seminiferous tubules, the function of the place for spermatogenesis will be directly impaired. If the process of spermatogenesis is blocked at the key stages of meiosis and sperm formation, the number of sperm will be sharply reduced or the quality of sperm will be seriously decreased, thereby directly leading to the decline of semen quality of breeding bulls, even causing infertility, and seriously affecting the economic benefits of beef cattle breeding. The process of spermatogenesis is regulated by a variety of cells in testis tissue, such as testicular Sertoli cells, interstitial cells and spermatogenic cells. Therefore, based on the core value of the reproductive performance of breeding bulls to beef cattle industry and the complexity and importance of the process of spermatogenesis, it is of important practical significance and application value to develop technical methods that can effectively improve the quality of semen of breeding bulls for promoting the sustainable development of beef cattle industry.

[0004] MSTRG.25151 is located on the X chromosome and is predicted to be a bovine Rhox homeobox family member 1 by NCBI BLASTN alignment analysis. Notably, the reproductive homeobox (RHOX) genes on the X chromosome were first identified in mice in the 1990s, and subsequent studies have confirmed their crucial role in the process of reproduction. Among various transcription factors with key regulatory roles, homeobox sequences encode "homeodomain" DNA-binding motifs. To date, 33 Rhox genes have been identified in the mouse genome. Maclean et al. found that disruption of Rhox5 in male mice increased male germ cell apoptosis and reduced sperm production, sperm motility and fertility. Tan et al. found that RHOX10 promotes mouse pro-spermatogonial cell (ProSG) differentiation through a conserved transcriptional cascade involving key germ cell TFs DMRT1 and ZBTB16 by using in vitro ProSG differentiation assay. Oh et al. found that Rhox8 KO adult male mice had lower fertility, disrupted spermatogenesis, increased germ cell apoptosis, reduced sperm number and motility, and clearly demonstrated the important role of Rhox8 in complete spermatogenesis and optimal male fertility. Compared with the mouse Rhox genes, the study of human RHOX genes is very limited. Yi et al. found that RHOX F1 was absent in patient testis biopsies, and histological analysis showed a phenotype supporting Sertoli cell syndrome, while RHOXF1 mutations significantly reduced the content of RHOX F1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V and p.R160X mutants of RHOX F1 caused increased accumulation of RHOX F1 in cytoplasmic granules, while the p.V130M and p.R160X mutants could disrupt downstream spermatogenesis by disrupting the regulation of doublesex and mab-3-related transcription factor 1 (DMRT1) promoter activity. It was clear that the mutated RHOX F1 was the pathogenic X-linked gene in human oligospermia and azoospermia. In summary, the study of the pathophysiology of RHOX genes may confirm the important role of the family in the process of reproduction and may help us better understand the various causes of infertility, thus providing theoretical support for solving related problems.

[0005] The Black Hereford is the first new germplasm of beef cattle successfully bred by somatic cell nuclear transfer in China, and has very high meat value. The reproductive ability of a breeding bull is an important economic trait, which directly affects the production performance and reproductive efficiency of a cow. Improving the reproductive ability of a breeding bull can improve the pregnancy rate and fetal quality of a cow, thereby improving the reproductive effect and accelerating the breeding process of the Black Hereford. Therefore, further optimization of the germplasm resources of the Black Hereford is of great significance for the further popularization of the breed. SUMMARY

[0006] In order to further study the function of the new gene MSTRG.25151 of the Black Hereford, the application provides a recombinant monoclonal antibody for the MSTRG.25151 coding protein of the Black Hereford, which is specific, has clear WB bands and no cross-reaction, and provides a tool for the function research and protein expression detection of the MSTRG.25151 gene.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme.

[0008] An antibody against the MSTRG.25151 protein, The heavy chain variable region (VH) thereof comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the light chain variable region (VL) thereof comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; or, The heavy chain variable region (VH) thereof comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the light chain variable region (VL) thereof comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; or, The heavy chain variable region (VH) thereof comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and the light chain variable region (VL) thereof comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. The amino acid sequence of the MSTRG.25151 protein is shown in SEQ ID NO: 1.

[0009] Preferably, the above-mentioned antibody against the MSTRG.25151 protein, The amino acid sequence of the heavy chain variable region thereof is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 21; or, The amino acid sequence of the heavy chain variable region thereof is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 23; or, The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 25.

[0010] In some embodiments, the antibody against MSTRG.25151 protein has the amino acid sequence of the heavy chain shown as SEQ ID NO: 26, and the amino acid sequence of the light chain shown as SEQ ID NO: 27.

[0011] In some embodiments, the antibody against MSTRG.25151 protein has the amino acid sequence of the heavy chain shown as SEQ ID NO: 28, and the amino acid sequence of the light chain shown as SEQ ID NO: 29.

[0012] In some embodiments, the antibody against MSTRG.25151 protein has the amino acid sequence of the heavy chain shown as SEQ ID NO: 30, and the amino acid sequence of the light chain shown as SEQ ID NO: 31.

[0013] The antibody can be a monomer or a Fab class antibody; the Fab class antibody is selected from Fab, Fab', (Fab')2, or scFv.

[0014] Preferably, the antibody is a chimeric antibody, and the constant region of the antibody is bovine-derived.

[0015] The present application also provides a nucleotide encoding the above antibody, an expression vector containing the encoding nucleotide, and a cell expressing the above antibody.

[0016] Preferably, the expression vector is selected from any one of pcDNA3.4++, pcDNA3.1(+), pcDNA4 / HisMax, pCMV-Tag2B, and pCHO1.0; and the most preferred expression vector is pcDNA3.4++; and the cell is selected from any one of HEK293 cell, HEK293T cell, HEK293F cell, CHO-K1 cell, and CHO-S cell; and the most preferred host cell is HEK293 cell.

[0017] In order to express and secrete the antibody into the extracellular, the nucleotide encoding the above antibody further comprises a signal peptide sequence that can be fused for expression. The amino acid sequence of the signal peptide is shown as SEQ ID NO: 32-37.

[0018] The present application provides a recombinant antibody precursor containing the above signal peptide.

[0019] In some embodiments, the recombinant antibody against MSTRG.25151 protein has a heavy chain precursor with an amino acid sequence as set forth in SEQ ID NO: 44, and a light chain with an amino acid sequence as set forth in SEQ ID NO: 45.

[0020] In some embodiments, the recombinant antibody against MSTRG.25151 protein has a heavy chain precursor with an amino acid sequence as set forth in SEQ ID NO: 46, and a light chain with an amino acid sequence as set forth in SEQ ID NO: 47.

[0021] In some embodiments, the recombinant antibody precursor against MSTRG.25151 protein has a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 48, and a light chain with an amino acid sequence as set forth in SEQ ID NO: 49.

[0022] In some embodiments, the nucleotide sequence encoding the above-mentioned recombinant antibody precursor, a nucleotide sequence of the heavy chain thereof is as set forth in SEQ ID NO: 38, and a nucleotide sequence of the light chain thereof is as set forth in SEQ ID NO: 39; or, a nucleotide sequence of the heavy chain thereof is as set forth in SEQ ID NO: 40, and a nucleotide sequence of the light chain thereof is as set forth in SEQ ID NO: 41; or, a nucleotide sequence of the heavy chain thereof is as set forth in SEQ ID NO: 42, and a nucleotide sequence of the light chain thereof is as set forth in SEQ ID NO: 43.

[0023] The above-mentioned antibody or recombinant antibody precursor can be used for detecting the content of MSTRG.25151 protein, and can realize rapid evaluation of the breeding performance of a bull.

[0024] The present application also provides a reagent and a kit prepared from the above-mentioned antibody or recombinant antibody precursor.

[0025] The present application has the following advantages: This invention is the first to prepare a specific antibody for Western blotting (WB) detection of the protein encoded by the novel Blackjack cattle gene MSTRG.25151, filling the technological gap of lacking a dedicated detection tool for the functional study of this novel gene. It provides core technical support for the functional analysis and regulatory mechanism exploration of the MSTRG.25151 gene, effectively advancing research progress in the field of molecular biology of Blackjack cattle. Simultaneously, this invention clarifies the key technical parameters for antigen design, immunization protocol, and antibody screening. The process is standardized and reproducible, ensuring stable acquisition of the target antibody. This antibody can be directly used for WB detection of the MSTRG.25151 protein in bovine testicular tissue, providing an efficient and reliable technical tool for research on bovine molecular breeding and reproductive-related disease mechanisms. Furthermore, through a dual screening strategy combining ELISA primary screening and WB secondary screening, this invention effectively eliminates non-specific antibodies, ensuring high antibody specificity, no extraneous band interference during WB detection, and accurate and reliable results. The purified antibody can be stored under mild conditions, requiring no special storage equipment, facilitating long-term storage, transportation, and large-scale application. Attached Figure Description

[0026] Figure 1 For Rhox homeobox family, member 1-like ( Bos taurus Gene region map; Figure 2 The results of sequence alignment between MSTRG.25151 and human RHOXF1; Figure 3 The results of the protein sequence alignment between MSTRG.25151 and mouse RHOXF1; Figure 4 WB detection for k4862, k4863, and k4864; Figure 5 IHC detection for k4862, k4863, and k4864; Figure 6 Western blot (WB) detection for B cell screening; Figure 7 IHC testing for #62, #68, #102, #40, #54, and #116; Figure 8 WB detection for #54-P16, #116-3P14, and #118-3P24; Figure 9 IHC testing for #54-P16, #116-3P14, and #118-3P24; Figure 10 Western blot (WB) detection of antibody SDT-1764-clone 118; Figure 11IHC assay for antibody SDT-1764-clone 118. Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0028] Example 1: Preparation of anti-MSTRG.25151 monoclonal antibody 1. Functional Analysis of MSTRG.25151 Testicular tissue was collected from healthy Blackjack bulls (males) with similar rearing environments. The testicular tissue samples were pretreated using the iST sample pretreatment kit (PreOmics, Germany). The samples were ground in liquid nitrogen, and an appropriate amount of sample was added to 50 μL of lysis buffer. The mixture was heated at 95°C and 1000 rpm for 10 min. After cooling to room temperature, trypsin digestion buffer was added, and the mixture was incubated at 37°C and 500 rpm for 2 h with shaking. The enzymatic digestion reaction was terminated by adding stop buffer. Peptide desalting was performed using the iSTcartridge kit, followed by elution with 2 × 100 µL of elution buffer. The eluted peptides were vacuum-dried and stored at -80°C. High-pH reversed-phase separation and low-pH nano-HPLC-MS / MS analysis (DDA qualitative library construction) were then performed. Raw data were merged and analyzed using Spectronaut X (Biognosys AG). Finally, DDA data were acquired.

[0029] Proteomic analysis of Blackjack bulls with varying reproductive capacities yielded the nucleotide sequence shown in SEQ ID NO: 50, designated as gene MSTRG.25151. Sequence alignment of this gene using NCBI BLASTN showed that this new gene is related to bovine (…). Bos taurus The mRNA sequence (XM_002699473.6) of the Rhox homeobox family member 1-like is 942 bases in length. The alignment range is from position 47 to 820, with an extremely high degree of matching (100% identical, no gaps), indicating that the two have strong homology.

[0030] See LOC782892 Rhox homeobox family, member 1-like ( Bos taurus ), the result is as follows Figure 1As shown: the gene (NC_037357.1) is located on the X chromosome, the position on the chromosome is 4294488..4296311, and the position of the new gene MSTRG.25151 on the chromosome is 4294610..4294851, which is a containing relationship. The MSTRG.25151 protein sequence (SEQ ID NO: 1) is aligned with the protein sequence of human RHOX F1, and the results are as follows Figure 2 As shown: the protein sequence of human RHOX F1 (NC_000023.11) is 184 in length, the aligned region is 94-148, and there is a certain sequence homology (49% identity, 67% similarity) in a specific region without gap interference. The MSTRG.25151 protein is aligned with the protein sequence of mouse RHOX F1, and the results are as follows Figure 3 As shown: the protein sequence of mouse RHOX F1 (NC_034589.1) is 217 in length, the aligned region is 138-178, and the protein has a certain sequence homology (41% identity, 68% similarity) in a specific region.

[0031] At the same time, through further exploration of the X chromosome region near the bovine Rhox homeobox family, member 1-like gene, it is found that the gene has similar conserved gene sites (AKAP14, NKAP, etc.) near the human and mouse Rhox F1 gene chromosome position. In summary, the MSTRG.25151 gene may play a key regulatory role in the process of bovine reproductive development (such as spermatogenesis, ovarian development, etc.) or other important physiological processes, similar to human and mouse Rhox F1 genes.

[0032] 2. Preparation of MSTRG.25151 recombinant protein The target sequence described by the amino acid sequence SEQ ID NO: 2 (denoted as CP-17E antigen) is codon-optimized by adding enzyme cutting sites, and then inserted into the HindIII and BamHI enzyme cutting sites of pET-28a vector after cutting with HindIII and BamHI, and transformed into E. coli competent cells to screen positive clones and extract positive plasmids.

[0033] The plasmid is transformed into BL21 (DE3) competent cells by heat shock method, then inoculated into LB medium, and the seed liquid is cultured and induced at 37°C, and the bacterial cells are collected for subsequent purification. After the bacterial cells are broken, the supernatant is collected, the target protein is purified by Ni column, the protein is concentrated by ultrafiltration, and is stored in PBS Buffer containing 3% trehalose. According to the immune requirements, it is divided into corresponding specifications and made into freeze-dried powder.

[0034] 3. Animal immunization New Zealand white rabbits were immunized with recombinant MSTRG.25151 protein, and the specific immunization scheme was as follows: Three New Zealand white rabbits were selected, numbered K4862, K4863 and K4864, and each rabbit was immunized five times. The first immunization and the second immunization were spaced three weeks apart, and the remaining immunizations were spaced two weeks apart. The last immunization was a booster immunization. The first immunization was 0.4 mg (0.5 mL) of recombinant protein mixed with complete Freund's adjuvant (0.5 mL), and was subcutaneously immunized at 0.2 mL / point x 5 points. The second, third and fourth immunizations were 0.2 mg (0.5 mL) of recombinant protein mixed with incomplete Freund's adjuvant (0.5 mL), and were subcutaneously immunized at 0.2 mL / point x 5 points. The booster immunization was intravenous injection of 0.4 mg (0.5 mL) of recombinant protein.

[0035] Ten days after the fourth immunization, 15 mL of serum was taken for coating immunogen for titer detection. When the titer met the serum dilution ratio of 1:64,000 and the OD value was >0.3, the titer was considered qualified. Protein A affinity purification and polypeptide or recombinant protein affinity purification (retaining flow-through) were used for 3 rabbits. The purified serum was used for immunohistochemical detection of bovine testis tissue and WB.

[0036] Table 1 ELISA detection results The serum titer results are shown in Table 1: at a dilution of 1:64,000, the OD value of K4862 was 0.1018, the OD value of K4863 was 0.1465, and the OD value of K4864 was 0.2379, indicating that these samples had strong antibody production capacity for CP-17E antigen.

[0037] The purified serum WB detection results are shown in Figure 4 : K4863 and K4846 both had clear target bands, while the band of K4862 was not obvious, approaching zero. The IHC detection results are shown in Figure 5 : only in the testis of sample 1952, nuclear staining was observed, among which K4864 was the strongest, and there were a large number of brown staining areas in the tissue, indicating that the expression level of the target protein in the sample was high, the cells were dense, and the staining signal was strong. In summary, rabbit No. K4864 was selected for the next step.

[0038] 4. B-cell panning, clone screening The rabbit spleen was used for single B cell plating (6 plates). The supernatant of 6P was detected by polypeptide ELISA, and positive clones were selected for the confirm stage. The clones in the confirm stage were verified by ELSIA again, and the positive clones were screened by WB and IHC. The WB detection results showed thatFigure 6 ), there were more than 30 positive clones of the target band. The IHC detection results showed that (Figure 6) Figure 7 ), except for negative and non-specific clones, there were three expression patterns: sperm cell positive, cytoplasmic and nuclear positive, represented by clones #62, #40; spermatogonia positive, cytoplasmic and nuclear positive, represented by clones #68, #54, #116, #118, #3; sperm and spermatocyte positive, cytoplasmic positive, represented by clones #102, #111. Among them, the 1952 sample immunized by #54, #68 and #62 could be seen under high magnification that the cell arrangement pattern of the seminiferous tubule was extensive and strong expression of the target protein, and the seminiferous tubule structure of the 1970, 1977, 1987 and 6733 samples was complete, but the expression intensity and cell localization of the target protein were significantly different. In summary, #54, #116, #118, #68 and #62 were selected for recombinant antibody preparation.

[0039] 5. Gene recombination, pairing identification In the recombination stage, HEK293 cells were transfected in small amounts, and the supernatant of each pair was detected by ELISA (supernatant and 200-fold dilution). The cell supernatant was detected by IHC and WB. The WB detection results showed that (Figure 7) Figure 8 ), the recombinant monoclonal #54-P16, #116-3P14, #118-3P24 of the three clones #54, #116, #118 were positive in WB detection, with clear target bands, and #118-3P24 was the strongest, while the other two clones #62, #68 had messy sequences and no positive monoclonal was obtained.

[0040] The IHC detection results showed that (Figure 8) Figure 9 ), the IHC staining patterns of the three clones were similar, and the tissue integrity of each sample was good, and the seminiferous tubule structure of the testis was clear. Under high magnification, it could be seen that the target protein in the 1952 sample was strongly expressed, and the brown signal was widely distributed in the spermatogenic cells and peritubular cells of the seminiferous tubule, and the staining intensity was high and the proportion of positive cells was large, indicating that the activity of the target protein in this sample was significant, while in the 1970, 1987, 6733 samples, it was followed, and the staining was concentrated in the spermatogenic cell group of the seminiferous tubule, and the staining was lighter in the peritubular area. The 1977 sample was the weakest, and the staining was mainly limited to a few cells in the seminiferous tubule, and the signal distribution was relatively dispersed. Among them, #118-3P24 was the strongest. In summary, #118-3P24 was selected for production and purification, and was named SDT-1764-clone 118.

[0041] 6. Production, purification and identification E. coliThe system expresses MSTRG.25151 full-length recombinant protein as an immunogen, mixes the immunogen protein with complete Freund's adjuvant (1:1) and emulsifies, and uses subcutaneous injection method to immunize 3 New Zealand white rabbits respectively; after two weeks, the immunogen is emulsified with incomplete Freund's adjuvant (1:1) for the second, third and fourth immunization; after four immunizations, blood is taken to determine the serum titer by gradient dilution by ELISA; when the ELISA titer is 1:64,000, the OD 450 The rabbit is subjected to a spleen resection operation.

[0042] The above rabbit spleen tissue is ground, and a small population of B cells that can efficiently secrete monoclonal antibodies is obtained through cell sorting and limiting dilution. The B cell population that secretes specific antibodies is screened through ELISA, IHC and WB, and the cDNA of the heavy and light chain gene sequences of the specific B cells is obtained through RT-PCR. In the molecular cloning stage, in the step of constructing a recombinant expression vector, different signal peptides (SEQ ID NO: 32-37) are selected according to the characteristics of the antibody amino acid sequence to splice with the coding sequences of the heavy chain variable region and the constant region, so that after protein synthesis, the protein can be guided to secrete outside the cell through the signal peptide, and the signal peptide can be specifically removed during protein secretion, which is convenient for subsequent purification; then the obtained cDNA is constructed into a pcDNA3.4++ vector through homologous recombination, and single colony sequencing is performed to obtain different heavy and light chain gene sequences.

[0043] The heavy and light chain gene sequences are all combined in pairs, and the constructed plasmids are transfected into 293 cell lines through transfection reagents, and the cell supernatant is collected. The collected 293 cell supernatant is verified through ELISA, IHC and WB to confirm that the antibodies expressed by the plasmids containing the nucleic acids in Table 2 have higher affinity to the antigen and have good immunoreaction.

[0044] Table 2 Coding sequences and amino acid sequences of #54, #116 and #118 antibody precursors Table 3 Variable region and CDR sequences of #54, #116 and #118 antibodies The expression vector of SDT-1764-clone 118 is subjected to a large number of cell transfections, and after 3-5 days of continuous culture, the cell suspension is collected, centrifuged, and the supernatant is taken. The cell supernatant is purified by Protein A column affinity chromatography to obtain an anti-MSTRG.25151 recombinant monoclonal antibody with a purity of more than 95%.

[0045] Example 2 Application of anti-MSTRG.25151 recombinant monoclonal antibody SDT-1764-clone 118 in detection of protein molecules IHC detection: Paraffin tissue chip of bovine testis tissue was placed in a drying oven to be baked at 63-65°C for about 1 hour. Dewaxing and hydration (all operations were completed in a fume hood) was performed by placing the slices in xylene twice, 100% alcohol twice, 90% alcohol twice, 75% alcohol once, and 50% alcohol once. After completion, the slices were removed and washed with distilled water three times. The slices were placed in a staining box containing sodium citrate buffer or EDTA repair solution, 2 L of distilled water was added to the antigen repair pot, and the staining box was placed in a pressure cooker for high temperature and pressure repair. After the pressure cooker started to release gas, the exhaust valve was closed and the heating was continued for 40 minutes. Then the heating was stopped, the inner pot was taken out, and it was naturally cooled and washed with distilled water. The slices were washed with 1xPBS three times. The antigen repaired slices were placed in 3% H2O2 solution for 10 minutes (operation in a fume hood), and then washed with 1xPBS three times. Non-specific antigens were blocked with 5% BSA + 10% goat serum (PBST preparation) for 30 minutes at room temperature. After blocking, the blocking solution on the slices was knocked off, and the diluted primary antibody was added, and incubated at 37°C for 1 hour. The next morning, the wet box was taken out and incubated at room temperature for 10 minutes, and then washed with 1xPBST three times. The secondary antibody (Anti-Rabbit and Mouse HRP-DAB IHC detection kit) was added and incubated at room temperature for 30 minutes, and then washed with PBST three times. 6 mL of color developing buffer was added to 6 drops of color developing solution, and fresh color developing solution was added to each slice. Color development was observed with the naked eye for 1-2 minutes, and the time was stopped by washing with tap water when brownish yellow color appeared. The slices were stained with hematoxylin for 2-5 minutes, washed with distilled water, and bleached in PBS for 30 seconds. The slices were dehydrated and dried in gradient alcohol (50% alcohol, 75% alcohol, 90% alcohol, 100% alcohol, three times, and xylene twice). The slices were mounted on glass slides by adding 50-100 μL of neutral balsam to each slice, and then slowly covering the glass slide. The mounted slices were dried overnight, and the next day the results of immunohistochemistry were observed under a microscope and scanned using a tissue section scanner. The results showed that the staining signals of the samples in 1952, 1970, 1987, etc. were clear, mainly concentrated in the cells of the testicular seminiferous tubules, and the staining patterns of different samples were consistent, indicating that the tissue specificity of the antibody was stable. Figure 10

[0046] ​WB detection: The corresponding film was carefully cut with a knife, and the marker was marked on the film. The film was soaked with 1xTBST, and the TBST was poured out. 5% skim milk powder / TBS solution was added, and the incubator was shaken for 1 hour at 40 r / min. During the blocking process, the antibody to be detected and the positive control were prepared and diluted according to the requirements. The incubation volume of the small culture dish was 10 mL, the incubation volume of the QC antibody incubation box was 5 mL, and the incubation volume of the cut strip plate was 2 mL. The diluted antibody in the incubation box and the cut strip plate was shaken on the shaker for 5 minutes at 40 r / min. After the blocking was completed, the surface of the blocked film was shaken dry and placed in the corresponding primary antibody. Incubate on the shaker for 1 h at 40 r / min. After the primary antibody incubation was completed, wash with TBST three times, and then wash with 1xTBST three times on the shaker at a speed of 85-100 r / min. The prepared secondary antibody was placed on the shaker and shaken for 10 minutes at a speed of 40 r / min. The film washed with the primary antibody was poured out with TBST cleaning solution, and the secondary antibody was added and incubated on the shaker for 1 h at a speed of 40 r / min. The secondary antibody was washed with 1xTBST three times, and then washed with 1xTBST three times at a speed of 100 r / min. Mix substrate A and substrate B at a ratio of 1:1, mix well, and restore to room temperature. Add about 1.5 mL of substrate mixture to each film. Different types of substrates have corresponding incubation times. Choose the appropriate incubation time according to the instructions. Then use a water-absorbing paper to absorb the excess substrate. Place the detected film on the sample platform, close the dark room door, open the exposure collection program, select the appropriate shooting distance, choose the shooting mode, set the exposure time, and turn on the white light imaging mode. The results are shown in (Figure 16) Figure 11 A clear band appeared in the lane, corresponding to a molecular weight of about 30 kDa. The band was single and had no obvious impurity band, indicating that the antibody SDT-1764-clone 118 could specifically recognize and bind to the target protein in the bovine testis tissue lysate, and the WB specificity was good.

[0047] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. An antibody against MSTRG.25151 protein, characterized in that, Its heavy chain variable region includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and its light chain variable region includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or, Its heavy chain variable region includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and its light chain variable region includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13; or, Its heavy chain variable region includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, and its light chain variable region includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; The amino acid sequence of the MSTRG.25151 protein is shown in SEQ ID NO:

1.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:20, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; or, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 22, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 23; or, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

25.

3. The antibody according to claim 1, characterized in that, Its heavy chain amino acid sequence is shown in SEQ ID NO: 26, and its light chain amino acid sequence is shown in SEQ ID NO: 27; or, The amino acid sequence of its heavy chain is shown in SEQ ID NO: 28, and the amino acid sequence of its light chain is shown in SEQ ID NO: 29; or, The amino acid sequence of its heavy chain is shown in SEQ ID NO: 30, and the amino acid sequence of its light chain is shown in SEQ ID NO:

31.

4. The antibody according to claim 1, characterized in that, The antibody is a monomer or a Fab-type antibody; the Fab-type antibody is selected from Fab, Fab', (Fab')2 or scFv.

5. The antibody according to claim 1, characterized in that, The antibody is a chimeric antibody, and the constant region of the antibody is of bovine origin.

6. A nucleotide encoding an antibody as described in any one of claims 1-5, an expression vector comprising the encoding nucleotide, and a cell expressing the antibody described above.

7. The expression vector and cell according to claim 6, characterized in that, The expression vector is selected from any one of pcDNA3.4++, pcDNA3.1 (+), pcDNA4 / HisMax, pCMV-Tag2B, and pCHO1.0; preferably pcDNA3.4++. The cells are selected from any one of HEK293 cells, HEK293T cells, HEK293F cells, CHO-K1 cells, and CHO-S cells; HEK293 cells are preferred.

8. The nucleotide according to claim 6, characterized in that, The nucleotide also includes a signal peptide sequence that can be fused for expression; preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 32-37.

9. A recombinant antibody precursor against MSTRG.25151 protein, characterized in that, The amino acid sequence of its heavy chain precursor is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain is shown in SEQ ID NO: 45; or, The amino acid sequence of its heavy chain precursor is shown in SEQ ID NO: 46, and the amino acid sequence of its light chain is shown in SEQ ID NO: 47; or, The amino acid sequence of its heavy chain is shown in SEQ ID NO: 48, and the amino acid sequence of its light chain is shown in SEQ ID NO:

49. Preferably, the nucleotide encoding the above-mentioned recombinant antibody precursor has the nucleotide sequence of its heavy chain as shown in SEQ ID NO: 38, and the nucleotide sequence of its light chain as shown in SEQ ID NO: 39; or, The nucleotide sequence of its heavy chain is shown in SEQ ID NO: 40, and the nucleotide sequence of its light chain is shown in SEQ ID NO: 41; or, The nucleotide sequence of its heavy chain is shown in SEQ ID NO: 42, and the nucleotide sequence of its light chain is shown in SEQ ID NO:

43.

10. The use of an antibody as described in any one of claims 1-5, or a recombinant antibody precursor as described in claim 9, in the detection of bovine MSTRG.25151 protein content, and the reagents and kits for its preparation.

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