A yak p62 protein polyclonal antibody and preparation method thereof

Abstract

The present invention discloses a yak p62 protein polyclonal antibody and a preparation method thereof, and belongs to the field of biotechnology. The present invention constructs a recombinant plasmid containing a yak p62 protein encoding gene, transforms Escherichia coli, induces the expression of a recombinant protein of yak p62, immunizes an animal after purification of the recombinant protein, and obtains a polyclonal antibody. The nucleotide sequence of the yak p62 protein encoding gene is shown in SEQ ID NO.3, and the amino acid sequence of the yak p62 recombinant protein is shown in SEQ ID NO.4. The preparation method of the present invention is short in time, convenient in operation, and low in cost. The prepared antibody has high titer and good specificity, which can not only meet the needs of yak-related molecular experiments, but also provide basic materials for subsequent research.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a yak p62 protein polyclonal antibody and a preparation method thereof. Background Art

[0002] Yak is a mammal of the genus Bovis in the family Bovidae of the order Artiodactyla. It is mainly distributed in plateau areas. As a special economic animal in alpine pastoral areas, it has become a dominant livestock in the plateau and an important source of income for herders due to its adaptability to special environments such as high cold and low oxygen. However, the natural reproduction rate of yaks is low. Therefore, the reproductive physiological activities of yaks have always been a hot topic of research.

[0003] Autophagy is a ubiquitous degradation pathway in eukaryotic cells that can remove damaged or aged organelles and invading pathogens, degrade long-lived proteins, maintain homeostasis in the intracellular environment, and also make adaptive adjustments to external environmental changes such as hunger, hormone imbalance, and oxidative stress. Studies have shown that autophagy is involved in many reproductive-related physiological and pathological processes, such as follicle development, fertilization, embryo implantation, inflammation, etc.

[0004] p62 (sequestosome1, SQSTM1) is a multifunctional ubiquitination-binding adaptor protein with a molecular weight of 62kDa. It is an important component of multiple protein complexes in cells and is widely recognized as an autophagy receptor protein. The p62 protein was originally discovered as a ligand for atypical protein kinase C (aPKC). With the deepening of research, scholars have found that the p62 protein can participate in a variety of biological functions in cells by interacting with many proteins, such as the ubiquitin protease system degradation system and the autophagy lysosome degradation system. p62 has multiple functional domains, and various signaling molecules (LC3, TRAF6, Keap1, Caspase8, etc.) bind to the domains to participate in autophagy, apoptosis, inflammation and other processes.

[0005] Antibodies are a type of biological molecule that plays an important role in the immune system. They are widely used in bioassay technology and in the diagnosis and treatment of diseases in humans and other animals. The preparation of polyclonal antibodies only requires the antigen to be directly injected into the animal for immunization. After multiple immunizations and ELISA testing of its titer, the serum is collected and purified to obtain polyclonal antibodies. Therefore, the preparation cycle of polyclonal antibodies is short and the preparation cost is low. In protein blotting, immunohistochemistry and immunofluorescence experiments, since polyclonal antibodies recognize multiple antigen epitopes, even if a few antigen epitopes are destroyed or the antigen conformation changes, the results of the experiment will not be affected. Under the same conditions, the use of polyclonal antibodies can improve the sensitivity of detection and make it easier to detect proteins with low abundance.

[0006] During the yak-related experiments, the scientific research progress was affected by the lack of yak-specific antibodies. p62 is a key protein in the physiological activities of yaks, and its expression characteristics in yak tissues have not been reported. Therefore, there is an urgent need for a technical solution for yak p62 protein polyclonal antibodies and the expression characteristics of p62 protein in yak tissues through immunohistochemistry and immunofluorescence techniques. Summary of the invention

[0007] The purpose of the present invention is to provide a yak p62 protein polyclonal antibody and a preparation method thereof to solve the problems existing in the above-mentioned prior art. The present invention successfully prepared an anti-p62 specific polyclonal antibody using recombinant p62 protein, which has a short preparation time, convenient operation, low cost, high titer and good specificity.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for preparing a yak p62 protein polyclonal antibody, comprising the following steps:

[0010] A recombinant plasmid containing a yak p62 protein coding gene is constructed, and after transformation into Escherichia coli, the recombinant protein of yak p62 is induced to express, the recombinant protein is purified and then used to immunize an animal to obtain immune serum, and the immune serum is purified to obtain the yak p62 protein polyclonal antibody;

[0011] The nucleotide sequence of the yak p62 protein encoding gene is shown in SEQ ID NO.3, and the amino acid sequence of the yak p62 recombinant protein is shown in SEQ ID NO.4.

[0012] Furthermore, the Escherichia coli is competent Escherichia coli BL21.

[0013] Furthermore, the specific steps of inducing the expression of the recombinant protein of yak p62 are: inoculating the transformed Escherichia coli into LB medium containing kanamycin for cultivation, and after amplification of the culture, adding IPTG solution to induce expression to obtain the recombinant protein of yak p62.

[0014] Furthermore, the concentration of kanamycin is 50 μg / mL.

[0015] Furthermore, the induction culture temperature is 37° C. and the culture time is 14 h.

[0016] Furthermore, the number of times of the amplification culture is 2 times. After the amplification culture, the bacterial OD 600nm It is 0.6-0.8.

[0017] Furthermore, the concentration of the IPTG solution is 1 mM.

[0018] Furthermore, after adding the IPTG solution, the expression was induced at 37° C. for 4 h.

[0019] Furthermore, the recombinant protein is purified by nickel column affinity enrichment.

[0020] The present invention also provides a yak p62 protein polyclonal antibody obtained by the preparation method.

[0021] The present invention discloses the following technical effects:

[0022] The present invention successfully prepared anti-p62 specific polyclonal antibodies using recombinant p62 protein. The antibody has a short preparation time, convenient operation, low cost, high titer and good specificity. It can not only meet the needs of yak-related molecular experiments, but also provide basic materials for subsequent research.

[0023] The present invention utilizes the prepared yak p62 protein polyclonal antibody to detect the expression characteristics of p62 in the yak oviduct and uterus for the first time through immunohistochemistry and immunofluorescence, verifies the effect of the antibody in practical application, and makes up for the lack of yak p62 polyclonal antibody and the lack of research on the expression characteristics of yak p62 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is the gel electrophoresis of yak p62 gene nucleic acid, where M: Marker; 1, 2: electrophoresis of p62 gene amplified using yak uterine tissue as template;

[0026] Figure 2 This is the gel electrophoresis of yak p62 recombinant plasmid nucleic acid, where M: Marker; 1: p62 recombinant plasmid electrophoresis, 2: electrophoresis after NdeI and BamHI digestion;

[0027] Figure 3 The results of SDS-PAGE of monoclonal induced expression of p62 protein, where M: Marker; 1: uninduced; 2-5: different induced clone strains;

[0028] Figure 4 This is the SDS-PAGE electrophoresis diagram of p62 protein purification, where M: Marker; 1: flow-through protein sample; 2-5: purified p62 protein:

[0029] Figure 5 This is the SDS-PAGE electrophoresis diagram of p62 final serum purification, where M: Marker; 1-6: p62 final serum purification;

[0030] Figure 6 These are the WB bands of yak p62 polyclonal antibody, where 1-2: yak oviduct tissue; 3-4: yak uterus tissue;

[0031] Figure 7 To detect the specificity of yak p62 polyclonal antibody by immunohistochemistry, EM: fallopian tube mucosal epithelium, EN: endometrial epithelium, UG: uterine gland;

[0032] Figure 8 Characterization of yak p62 polyclonal antibody for immunofluorescence detection. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] In the present invention, the yak uterus and oviduct tissue samples were collected from a slaughterhouse in Xining, Qinghai Province, China; New Zealand white rabbits were provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; and the Escherichia coli vector plasmid pET-28a (+) and BL21 (DE3) competent cells were provided by KMD Biotechnology (Tianjin) Co., Ltd. Unless otherwise specified, other materials and reagents can be purchased through conventional commercial channels.

[0039] Example 1

[0040] 1. Experimental Methods

[0041] 1. Cloning of yak p62 gene

[0042] Total RNA was extracted from yak tissue samples, and cDNA was obtained after reverse transcription and stored at -20°C.

[0043] Using NCBI cattle (Bos taurus) p62 gene (NM_176641.1) as the reference sequence, primers were designed using primerprimer5. The primer sequences were: Forward: GGACCGGGAAGTAGCT (SEQ ID NO.1), Reverse: CACTGTTCCTCCGAGTTAA (SEQ ID NO.2), Tm: 56, and product length: 1517 bp.

[0044] The reverse transcribed cDNA was used as a template to amplify the yak p62 gene. The PCR reaction system was: MasterMixes 10μL, cDNA 2μL, primers 0.5μL each, ddH2O 7μL. PCR reaction program: 98℃, 3min; 95℃, 30s; 58℃, 30s; 72℃, 2min; 72℃, 5min. Product specificity was verified by agarose gel electrophoresis.

[0045] The correct product was recovered by referring to the universal DNA gel recovery kit (Tiangen, DP214), connected to the pM19-T (Takara, 6013) vector, and transformed into DH5α competent cells (Takara, 9057). In 100mL of agar medium, 500μL of X-gal solution (20mg / mL), 250μL of IPTG solution (50mg / mL) and 100μL of Amp solution (ampicillin 100mg / mL) were added to make a plate culture medium containing X-Gal, IPTG and Amp. The competent cells were coated on this solid culture medium and cultured overnight (12h-14h), and the monoclonal positive white colonies were picked and added to ddH2O as a template. After PCR verification, they were placed in liquid LB culture medium, cultured overnight, and the bacterial liquid system was expanded and sent to Shanghai Biotech for sequencing verification.

[0046] 2. Construction of yak p62 gene recombinant plasmid

[0047] The correctly sequenced sequence was synthesized by Beijing KAMD Biotechnology Co., Ltd. (containing NdeI and BamHI restriction sites), and the pET-28a(+) plasmid was cut using endonucleases NdeI (Takara, 1621) and BamHI (Takara, 1605). The synthesized yak p62 gene was connected to the pET-28a(+) plasmid and transformed into DH5α competent cells. After overnight culture, a single clone was picked and verified by PCR. The plasmid was extracted with reference to the plasmid mini-extraction kit (Tiangen, DP103), and verified by nucleic acid gel electrophoresis after enzyme digestion. The rest was stored at -20°C.

[0048] 3. Expression of yak p62 recombinant protein

[0049] Mix 10 μL of the stored yak p62 recombinant plasmid with 50 μL of BL21 (DE3) competent cells, incubate on ice for 30 min, incubate at 42°C for 90 s and then incubate on ice for 5 min. Take 30 μL and apply it to LB medium (containing 50 μg / mL kanamycin), incubate at 37°C for 14 h, pick a single clone and add 2 mL of LB liquid medium (containing 50 μg / mL kanamycin) to activate the cells until the OD 600nm After the cell OD reaches 0.6-0.8, transfer to 5 mL LB liquid medium (containing 50 μg / mL kanamycin) and culture until the cell OD 600nm After the p-value was 0.6-0.8, 1 mM IPTG solution was added, and the bacteria were collected after inducing expression at 37°C for 4 hours. The bacteria samples before and after induction were taken for SDS-PAGE analysis.

[0050] 4. Purification of yak p62 recombinant protein

[0051] Select the BL21 strain with good expression of the target protein, inoculate it into 1L LB medium (containing 50μg / mL kanamycin), and expand the culture at 220r / min and 37℃ to OD 600nm The pH value was 0.6-0.8, and then IPTG was added at a final concentration of 1 mM. After induction culture at 30°C for 12 h, the bacteria were collected by centrifugation at 4000 r / min for later use. The collected bacteria were resuspended in 40 mL of a mixed solution of 20 mM phosphate buffer (PB) + 150 mM sodium chloride (NaCl) (pH 7.2), and ultrasonically broken in an ice bath for 3 s, 5 s intervals, and continued for 15 min; centrifuged at 12000 r / min, 4°C for 20 min, and the supernatant and precipitate were collected.

[0052] The obtained precipitate was washed once with 20mM PB+150mM NaCl (pH8.0) buffer and once with 20mM PB+150mM NaCl+0.3% TritonX-100 (pH8.0) buffer. Then, gradient elution was performed using 20mM PB+150mM NaCl buffer (pH8.0) containing different urea (Urea) concentrations (1M, 2M, 3M, 4M). The precipitate was dissolved with 15mL of 20mM PB+150mMNaCl+8M Urea (pH8.0) buffer, centrifuged, and the precipitate was re-dissolved and mixed with the supernatant, filtered through a 0.22μm filter membrane, and then purified by affinity enrichment using a nickel (Ni) column. (Equilibrium buffer: 20mM PB+150mM NaCl+8M Urea, pH8.0; Cleaning buffer: 20mM PB+150mM NaCl+8M Urea+5mM imidazole, pH8.0; 20mM PB+150mM NaCl+8M Urea+20mM imidazole, pH8.0; 20mM PB+150mM NaCl+8MUrea+50mM imidazole, pH8.0; 20mM PB+150mM NaCl+8M Urea+80mM imidazole, pH8.0; Elution buffer: 20mM PB+150mM NaCl+8M Urea+300mM imidazole, pH8.0.) Flow through, purify the sample, and perform SDS-PAGE analysis after denaturation.

[0053] 5. Animals immunized with yak p62 recombinant protein

[0054] Purified yak p62 recombinant protein was used to immunize a New Zealand white rabbit; immunization was performed at 0, 2, 3, 4, and 5 weeks, through multiple subcutaneous inoculations on the back; immunization dose: 1.0 mL / rabbit for the first injection, Freund's complete adjuvant and the sample were mixed and emulsified in equal volumes; 2-5 injections, 0.5 mL / rabbit, Freund's incomplete adjuvant and the sample were mixed and emulsified in equal volumes. Blood was collected 1 week after the 4th injection, and the serum titer was tested by ELISA. Blood was collected after the 5th booster injection to separate the serum for antibody purification.

[0055] 6. ELISA to detect the titer of rabbit polyclonal antibody serum

[0056] The yak p62 recombinant protein was diluted to 1.0 μg / mL with 0.01M phosphate buffer and coated on a 96-well ELISA plate at a dilution of 1:100 at 4°C overnight. The plate was washed three times with 0.01M phosphate buffer containing 0.05% Tween-20, and blocked with phosphate buffer containing 10% fetal bovine serum at 37°C for 2 h. The remaining blocking solution in the wells was removed before use. Immunized rabbit serum and negative control were added in sequence for 10 2 -10 7 Add 100 μL of the diluted sample to each well of the 96-well plate, incubate at 37°C for 1 hour, wash three times with 0.01M PBST, add goat anti-rabbit IgG HRP and incubate at 37°C for 1 hour, wash five times, pat dry, add color substrate and color at 37°C for 10 minutes, stop with 2M H2SO4, and read at a wavelength of 450nm. Standard: At the same dilution multiple, the sample OD 450 If the absorbance value of the negative control is ≥ negative control × 2.1, it is judged as positive (if the absorbance value of the negative control is <0.05, it is calculated as 0.05).

[0057] 7. Purification of yak rabbit polyclonal anti-p62 serum

[0058] Clean the column, take an appropriate amount of protein G-Sepharose ethanol suspension, let it stand for gel precipitation, then remove the upper ethanol, and then suspend it several times with binding buffer to thoroughly wash away the ethanol. Mix the binding buffer with protein A / G into a 50-70% slurry and pour it into the column. Connect a peristaltic pump and load the column at a rate of 50cm / h. After the column is completely settled, continue to wash the column with 5-10 times the volume of binding buffer. Centrifuge the rabbit serum sample at 12,000×g for 10min, take the supernatant, filter it with a pore size of 0.45μm, and add it to the affinity column at a flow rate of 0.4mL / min. After entering the medium, adjust the flow rate down to 0.7mL / min. Keep the rate unchanged and collect the eluate in 2mL elution fractions. Add 0.1mL of neutralization buffer to the collection tube. Detect the A280 of each tube, stop elution when A280<0.05, and collect the part with A280≥0.2. The purified yak protein was added to 5× loading buffer at a ratio of 4:1, denatured at 100°C for 10 min, and the antibody purity was detected by SDS-PAGE electrophoresis.

[0059] 8. Specificity of yak p62 polyclonal antibody detected by Western blotting

[0060] Yak tissue total protein was extracted using high-efficiency RIPA lysis buffer (tissue / cell) (Solebo, R0010), 4× protein loading buffer (Solebo, P1015) was mixed with protein at a ratio of 1:3, denatured at 100°C for 10 min, and stored at -20°C. 8% gel was prepared, 10 μL of sample was loaded for SDS-PAGE electrophoresis, 0.45 μm pore size PVDF membrane was used, 110V, membrane transfer was performed for 65 min, 5% skim milk was shaken for 3 h, yak p62 polyclonal antibody was diluted 1:1000, and incubated at 4°C overnight. PBST was used to wash 6 times, 5 min each time; goat anti-rabbit HRP was diluted 1:8000, incubated at room temperature shaken for 50 min, and PBST was used to wash 6 times, 10 min each time. Expose and take pictures.

[0061] 9. Immunohistochemical detection of p62 expression characteristics in yak oviduct and uterus

[0062] The fixed tissue was removed from 4% paraformaldehyde, punched, embedded, sliced, and dried for 4 hours. The slices were dewaxed with alcohol, hydrated, and antigen repaired with citrate. The slices were washed with PBS, and immunohistochemistry experiments were performed according to the SP immunohistochemistry detection kit (Boosen, SPS-0023). 5% H2O2 solution was added and incubated at 37°C for 10 minutes. The slices were washed with PBS three times, and blocking solution (liquid A) was added and incubated at room temperature for 15 minutes. The primary antibody (1:100) was added and incubated at 4°C overnight. For the negative control, PBS was added. The slices were washed with PBS three times, and the secondary antibody (liquid B) was added and incubated at 37°C for 15 minutes. The slices were washed with PBS three times, and the tertiary antibody (liquid C) was added and incubated at 37°C for 15 minutes. The slices were washed with PBS three times, and DAB was used for color development. The reaction was terminated with tap water. The slices were washed with distilled water for 5 minutes, and hematoxylin was used for counterstaining for 3 minutes. The slices were dehydrated, transparentized, sealed with resin, and photographed.

[0063] 10. Immunofluorescence detection of p62 expression characteristics in yak oviduct and uterus

[0064] The fixed tissue was removed from 4% paraformaldehyde, punched, embedded, sliced, and dried for 4 hours. The slices were dewaxed with alcohol, hydrated, and antigen repaired with citrate. The slices were washed 3 times with PBS, permeabilized with 0.5% TritonX-100 for 10 minutes, washed 3 times with PBS, and 5% H2O2 solution was added at 37°C for 10 minutes; washed 3 times with PBS, and 5% BSA was added at room temperature for 2 hours; p62 polyclonal antibody (1:100) was added at 4°C overnight, washed 3 times with PBS, and fluorescent secondary antibody (1:1000) was added at 37°C for 1 hour; washed 3 times with PBS, DAPI nuclear staining for 3 minutes, washed 3 times with PBS, anti-fluorescence quencher was added, the slices were sealed, and photographed with a laser confocal microscope.

[0065] 2. Results and Analysis

[0066] 1. Cloning of yak p62 gene

[0067] After PCR amplification, the agarose verification bands were clear and single, such as Figure 1 The product was connected with pM19-T and transformed into DH5α cells. The bacterial solution was sent to Shanghai Bioengineering for sequencing. The result was correct and has been submitted to NCBI Genbank. The accession number is OR450919.1. The specific sequence is as follows:

[0068] p62 gene nucleotide sequence (SEQ ID NO.3):

[0069]

[0070] p62 protein amino acid sequence (SEQ ID NO.4):

[0071] MASLTVKAYLLGKEDAAREIRRFSFCFSPEPEAEAEAAPGSRPCERLLSRVAALFPVLRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRRDQR PSCAQEVPRNMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCSACEGKGLHREHGKLAFPSPIGHFSEGFSHSRWLRKLKHGHFGWPAWEMGTPGNWSPRPPQAGDAHPGP ATESASGPPEDPSVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPTSAGSSSTEEKCSSQPSSCCSDPSKPDRDVEGTAQSLTEQMNKIALESGGQLEEQMESDN CSGGDDDWTHLSSKEVDPSTGELQSLQMPESEGPSSLDPSQEGPTGLKEAALYPHLPPEADPRLIESLSQMLSMGFSDEGGWLTRLLQTKNYDIGAALDTIQYSKHPPPL.

[0072] 2. Construction of yak p62 recombinant plasmid

[0073] The amplified p62 gene sequence was connected with the pET-28a(+) plasmid and transformed into DH5α cells. The bacterial solution was sequenced, the plasmid was extracted, and the gel electrophoresis results were as follows: Figure 2 As shown, it indicates that the plasmid was successfully constructed.

[0074] 3. Expression of yak p62 protein

[0075] The yak p62 recombinant plasmid was transformed into BL21(DE3) competent cells, and a single clone was picked and cultured until the bacterial OD 600nm After the concentration of IPTG solution was 0.6-0.8, the final concentration of IPTG solution was added, and the cells were collected after induction at 37℃ for 4 hours. The SDS-PAGE of the cells before and after induction was as shown in Figure 3 As shown, it indicates that the induced bacteria express yak p62 recombinant protein.

[0076] 4. Purification of Yak p62 Recombinant Protein

[0077] Select the BL21 strain that expresses the target protein better, expand the culture system, and OD 600nmThe final concentration of IPTG was 0.6-0.8, and the final concentration of 1mM IPTG was added for induction culture at 30℃ for 12h. The cells were collected by centrifugation, resuspended with a mixed solution of 20mM PB+150mM NaCl (pH7.2), broken, and the supernatant and precipitate were collected by centrifugation. The precipitate was dissolved with 15mL of 20mM PB+150mM NaCl+8M Urea (pH8.0) buffer, centrifuged, and the precipitate was re-dissolved and mixed with the supernatant, filtered through a 0.22μm filter membrane, and then purified by nickel (Ni) column affinity enrichment. The flow-through, purified sample, and SDS-PAGE analysis were performed after denaturation. The results are as follows Figure 4 As shown, the purified protein band was single and had good specificity. Finally, the yak p62 recombinant protein was reconstituted with 0.9% saline to 1 mg / mL as an immunogen for rabbit polyclonal antibody preparation and screening.

[0078] 5. Preparation of polyclonal antibodies

[0079] (1) Preparation and titer detection of yak p62 antiserum

[0080] Purified yak p62 protein was used to immunize New Zealand white rabbits, blood was collected, and serum was obtained by centrifugation. The serum titer was tested by ELISA. The results are shown in Table 1. The titer of anti-yak p62 serum was 1:10. 5 . Titer evaluation criteria: Under the same dilution multiple, the sample OD450 value ≥ negative control × 2.1. If the negative control absorbance value is less than 0.05, it is calculated as 0.05 to determine the positive standard.

[0081] Table 1 ELISA test serum titer results

[0082]

[0083] (2) Purification of yak p62 polyclonal antiserum

[0084] The yak p62 antiserum was purified by Staphylococcus protein A (SPA) method. The SDS-PAGE results after purification were as follows: Figure 5 The titer results of ELISA are shown in Table 2. The titer of yak p62 polyclonal antibody is 1:10 6 .

[0085] Table 2 ELISA test results of purified serum titer

[0086]

[0087] 6. Specificity verification of yak p62 polyclonal antibody

[0088] The specificity of the prepared yak p62 antibody was detected by WB using yak oviduct and uterine tissue. The results are as follows Figure 6As shown in the figure, the band is single and has good specificity. The results of antibody specificity detection by immunohistochemistry and immunofluorescence are shown in the figure. Figure 7 and 8 As shown, p62 protein is localized in the mucosal epithelium, uterine glands, and endometrium of the yak oviduct, and is expressed in the cytoplasm.

[0089] The invention successfully constructs a yak p62 recombinant plasmid, expresses and purifies a yak p62 recombinant protein, immunizes New Zealand white rabbits with the recombinant p62 protein, collects serum, and purifies the yak p62 protein polyclonal antibody, which has immunogenicity.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a polyclonal antibody against yak p62 protein, It is characterized in that The following steps are involved: A recombinant plasmid containing a yak p62 protein coding gene is constructed, and after transformation into Escherichia coli, the recombinant protein of yak p62 is induced to express, and the recombinant protein is purified and then used to immunize New Zealand white rabbits to obtain immune serum, and the immune serum is purified to obtain the yak p62 protein polyclonal antibody; The nucleotide sequence of the yak p62 protein encoding gene is shown in SEQ ID NO.3, and the amino acid sequence of the yak p62 recombinant protein is shown in SEQ ID NO.

4.

2. The preparation method according to claim 1, It is characterized in that The Escherichia coli is competent Escherichia coli BL21.

3. The preparation method according to claim 1, It is characterized in that The specific steps of inducing the expression of the recombinant protein of yak p62 are: inoculating the transformed Escherichia coli into LB medium containing kanamycin for cultivation, and after amplification of the culture, adding IPTG solution to induce expression to obtain the recombinant protein of yak p62.

4. The preparation method according to claim 3, It is characterized in that The concentration of kanamycin is 50 μg / mL.

5. The preparation method according to claim 3, It is characterized in that The culture temperature is 37° C. and the culture time is 14 h.

6. The preparation method according to claim 3, It is characterized in that The number of times of the amplification culture is 2 times. After the amplification culture, the bacterial OD 600nm It is 0.6-0.

8.

7. The preparation method according to claim 3, It is characterized in that The concentration of the IPTG solution is 1 mM.

8. The preparation method according to claim 3, It is characterized in that After adding the IPTG solution, the expression was induced at 37° C. for 4 h.

9. The preparation method according to claim 1, It is characterized in that The recombinant protein was purified by nickel column affinity enrichment.

10. The yak p62 protein polyclonal antibody obtained by the preparation method according to any one of claims 1 to 9.

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