Preparation method and application of chicken P4HB protein polyclonal antibody

Abstract

The invention discloses a preparation method and application of a rabbit anti-chicken P4HB protein polyclonal antibody, including amplifying the chicken P4HB gene by a PCR method, homologously recombining it into a pCOLDII vector, and constructing a prokaryotic recombinant expression plasmid pCOLDII-P4HB. The recombinant plasmid is transformed into E.coli BL21 (DE3) and induced to express by IPTG, and the protein is purified by Ni-NTA affinity chromatography. After the purified HIS-P4HB protein is mixed and emulsified with an equal volume of Freund's complete adjuvant, the purified protein is immunized into New Zealand white rabbits, and multiple subcutaneous injections are performed on the back of the neck. The antibody is purified by ProteinA+G affinity chromatography and frozen in a refrigerator at 80°C. The prokaryotic expressed HIS-P4HB protein is expressed in the supernatant of competent cells, and the molecular weight is about 25KDa; after the experimental rabbit is immunized with P4HB recombinant protein 4 times, blood is collected and serum is separated. After purification, the antibody can identify the expression and cellular localization of native conformation and linearized P4HB protein by Western blot and IF.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry and molecular immunology, and specifically relates to a preparation method of a chicken P4HB protein polyclonal antibody and an application thereof. Background Art

[0002] Chicken P4HB protein, also known as Protein Disulfide Isomerase (PDI), is a protein widely found in chickens. It is mainly found in the endoplasmic reticulum (ER) of cells and can also be released to the cell surface and extracellular matrix. Its biological function is to promote the formation, rupture and rearrangement of disulfide bonds (SGS). SGS is a covalent bond connecting two cysteine ​​residues and is an important component for maintaining the spatial structure of proteins. Its changes can affect the structure and function of proteins. Studies have shown that P4HB plays the role of oxidoreductase, isomerase and molecular chaperone in the process of protein production and release. In addition, P4HB protein plays a key role in protein folding and functional modification. It can help proteins fold correctly and form functional structures, which is essential for maintaining protein homeostasis in cells. P4HB protein is also involved in regulating cell and cell signaling processes. This is of great significance for maintaining intercellular communication and coordinating cell behavior.

[0003] Since the discovery of protein disulfide isomerase, domestic and foreign researchers have conducted a lot of research on its structure and biological function, and found that it is closely related to thrombotic diseases, tumors and neurodegenerative diseases, suggesting that it has the potential to be a new therapeutic target and a biomarker for disease diagnosis and prognosis. As an important economic livestock and the main source of animal protein for people in my country, ensuring the health of chickens and improving their production performance is an important responsibility of animal husbandry and veterinary work. At the same time, as an important model animal, the research on chickens also provides important reference value for further research on human diseases. However, the lack of antibodies for detecting egg white in the market has seriously restricted the research on the function of chicken P4HB protein. Therefore, it is particularly important to prepare antibodies for the detection of chicken P4HB protein, which can be used to study the physiological function of P4HB in chickens and make up for the demand for this antibody in the market. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a chicken HIS-P4HB modified polypeptide.

[0007] In order to solve the above technical problems, the present invention provides the following technical solution: the amino acid sequence of the chicken HIS-P4HB modified polypeptide is shown in SEQ ID NO.3.

[0008] The chicken HIS-P4HB modified polypeptide is obtained by screening a peptide segment of the chicken P4HB polypeptide shown in SEQ ID NO.1, performing histidine tag modification, and then expressing it in prokaryotic cells.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide a polyclonal antibody against a chicken HIS-P4HB modified polypeptide, wherein the polyclonal antibody against a chicken HIS-P4HB modified polypeptide is obtained by immunizing the chicken HIS-P4HB modified polypeptide as an antigen.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a polyclonal antibody against a chicken HIS-P4HB modified polypeptide.

[0011] In order to solve the above technical problems, the present invention provides the following technical solutions: PCR amplification of chicken P4HB gene, and homologous recombination of the gene into an expression vector to construct a prokaryotic recombinant expression plasmid pCOLDII-P4HB;

[0012] The recombinant plasmid was transformed into E.coli BL21 and induced for expression with IPTG, and then the protein was purified by Ni-NTA affinity chromatography. The purified HIS-P4HB protein was mixed with an equal volume of Freund's complete adjuvant for emulsification and then immunized and the serum was separated. After purification by ProteinA+G affinity chromatography, it was frozen at -80°C to obtain a polyclonal antibody.

[0013] As a preferred embodiment of the preparation method of the present invention, the immunization conditions are multiple subcutaneous injections at the back of the neck, and the second and third immunizations are respectively performed at the third and fifth weeks after the first immunization, both of which are emulsified with Freund's incomplete adjuvant, and the method and dosage are the same as the first immunization.

[0014] As a preferred embodiment of the preparation method of the present invention, the immunization dose is 200 μg / mouse.

[0015] As a preferred embodiment of the preparation method of the present invention, blood is collected from the ear vein on the 9th day after the third immunization to separate serum. The fourth immunization is performed on the 7th week after the first immunization, and blood is collected from the heart one week later.

[0016] As a preferred embodiment of the preparation method of the present invention, the prokaryotic recombinantly expressed HIS-P4HB protein is expressed in the supernatant of E.coli BL21 cells, and has a molecular weight of about 25 KDa.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a polyclonal antibody against a chicken HIS-P4HB modified polypeptide for use in detecting chicken P4HB protein expression and studying the function and mechanism of action of the P4HB protein.

[0018] As a preferred embodiment of the application of the present invention, the polyclonal antibody is used to identify the expression and cellular localization of endogenous P4HB protein expressed by primary chicken macrophages by Western blot and indirect immunofluorescence.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention amplifies the chicken P4HB gene by PCR, and homologously recombines it into the pCOLDII vector to construct a prokaryotic recombinant expression plasmid pCOLDII-P4HB. The recombinant plasmid is transformed into E. coli BL21 (DE3) and induced to express with IPTG. The expressed HIS-P4HB recombinant protein is identified by SDS-PAGE method, and the SDS-PAGE result shows a clear band at about 25KDa. Then, the protein is purified by Ni-NTA affinity chromatography method, and the purified HIS-P4HB protein is mixed and emulsified with an equal volume of Freund's complete adjuvant, and then the purified protein is immunized into New Zealand white rabbits by multiple subcutaneous injections at the back of the neck, with an immunization dose of 200μg / rabbit. The second and third immunizations are performed at the third and fifth weeks after the first immunization, respectively, and both are emulsified with Freund's incomplete adjuvant, and the method and dose are the same as the first immunization. On the 9th day after the third immunization, blood is collected from the ear vein to separate serum. The fourth immunization is performed at the 7th week after the first immunization, and blood is collected from the heart one week later. The antibody was purified by ProteinA+G affinity chromatography and stored in a -80°C refrigerator. The expression and cellular localization of endogenous P4HB protein expressed by primary chicken macrophages (HD11) were identified by Western blot and indirect immunofluorescence staining (IF) to verify its application.

[0021] (2) The prokaryotically expressed HIS-P4HB protein of the present invention is expressed in the supernatant of E. coli BL21 cells, and has a molecular weight of about 25 KDa; after the experimental rabbits were immunized with P4HB recombinant protein four times, blood was collected and serum was separated. After purification, the antibody can be used for Western blot and IF to identify the expression and cellular localization of chicken endogenous P4HB protein.

[0022] (3) The present invention predicted antigenic determinants, used prokaryotic expression technology to prepare HIS-P4HB polypeptide, immunized New Zealand white rabbits, and successfully prepared its polyclonal antibody. The antibody was verified to be applicable to the qualitative, quantitative and cellular localization of chicken P4HB linear or native conformation proteins, which laid a foundation for further exploring the biological function of chicken protein disulfide isomerase and the biological function of the catalytic reaction produced by the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. 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 creative labor. Among them:

[0024] Figure 1 The results of the analysis of the hydrophilicity, antigenic index and surface accessibility of the chicken P4HB protein sequence using the bioinformatics software DNAstar (Protean) in Example 1 of the present invention.

[0025] Figure 2 This is an agarose gel electrophoresis diagram of the PCR amplified P4HB fragment in Example 2 of the present invention.

[0026] Figure 3 This is an agarose electrophoresis identification diagram of the bacterial solution after PCR in Example 2 of the present invention.

[0027] Figure 4 This is a SDS-PAGE result diagram of the expression position of the P4HB recombinant protein in Example 2 of the present invention.

[0028] Figure 5 This is a diagram showing the purification effect of the HIS-P4HB recombinant protein in Example 2 of the present invention.

[0029] Figure 6 This is the purification curve of P4HB polyclonal antibody in Example 2 of the present invention.

[0030] Figure 7 This is a titer chart of unpurified anti-P4HB serum and purified P4HB polyclonal antibody detected by indirect ELISA method in Example 2 of the present invention.

[0031] Figure 8 This is a diagram showing the results of a Western Blot test using different concentrations of P4HB recombinant protein to verify the specificity of the rabbit anti-chicken P4HB polyclonal antibody of the present invention in Example 2 of the present invention.

[0032] Fig. 9This is a graph showing the results of Western Blot detection of the expression level of chicken P4HB protein (linear structure) in the chicken cell line HD11 using the rabbit anti-chicken P4HB polyclonal antibody of the present invention in Example 2 of the present invention.

[0033] Fig.10 This is a graph of the IF results of using the rabbit anti-chicken P4HB polyclonal antibody of the present invention to detect the expression and localization of endogenous P4HB protein (native conformation) in the chicken HD11 cell line in Example 2 of the present invention.

[0034] Fig.11 This is a graph showing the results of Western Blot detection of the expression level of chicken P4HB protein (linear structure) in chicken liver using the rabbit anti-chicken P4HB polyclonal antibody of the present invention in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0036] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0037] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0038] Unless otherwise specified, the raw materials used in the examples of the present invention are commercially available.

[0039] Among them, Prime STAR Max DNA Polymerase was purchased from Baori Bioengineering Co., Ltd.; homologous recombination kit was purchased from Vazyme; DMEM medium and fetal bovine serum were purchased from Gibco; plasmid mini-extraction kit and gel recovery kit were purchased from OMEGA; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; infrared fluorescent labeled goat anti-rabbit was purchased from abcam, UK. Tris, glycine, isopropyl-β-D-thiogalactoside (IPTG), and dithiothreitol (DTT) were purchased from Beijing Solebao Technology Co., Ltd. Imidazole, Sigma-Aldrich, Merck KGaA, Germany; QuickBlue rapid staining solution, goat anti-rabbit-HRP rabbit were purchased from Suzhou Biolong Technology Co., Ltd.; BSA, Shanghai Yuanye Biotechnology Co., Ltd.; sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), disodium hydrogen phosphate·dihydrate (NaH2PO4·2H2O), disodium ethylenediaminetetraacetic acid·dihydrate (Na2EDTA·2H2O), sodium bicarbonate (NaHCO3), ethanol, magnesium chloride (MgCl), potassium chloride (KCl), Sinopharm Chemical Reagent Co., Ltd. Protein Marker (250μL), Suzhou Xinsaimei Biotechnology Co., Ltd. HD11 cells were stored in the laboratory; E.coli TOP10 competent cells and E.coliBL21 (DE3) competent cells were purchased from Shanghai Shenggong Biological Co., Ltd.; 2-month-old female New Zealand white rabbits were purchased from Jiangsu Academy of Agricultural Sciences.

[0040] Example 1

[0041] (1) Construction of recombinant plasmid:

[0042] Based on the chicken P4HB polypeptide sequence (SEQ ID NO.1), the hydrophilicity, surface accessibility, antigenic index and homology of its protein sequence were analyzed using the Protean module in DNAstar software, and a suitable peptide sequence (123aa-354aa) (SEQ ID NO.2) was screened out as the target sequence ( Figure 1 ), modified with a histidine tag, and expressed in prokaryotic cells to obtain the final chicken HIS-P4HB modified polypeptide sequence shown in SEQ ID NO.3. Figure 1 Implementation:

[0043] Based on the chicken P4HB polypeptide sequence, the hydrophilicity, surface accessibility, antigenic index and homology of its protein sequence were analyzed using the Protean module in the DNAstar software, and suitable peptide sequences (123aa-354aa) were screened as target sequences for histidine tag modification ( Figure 1). It is expected to improve the specific recognition ability of the prepared antibody to the antigen. The primers designed for the cDNA sequence corresponding to this region (synthesized by Beijing Qingke Biotechnology Co., Ltd., the expected PCR product size is 732 bp) are shown in Table 1.

[0044] Table 1

[0045] 5'-3' P4HB-pCOLDII recombinant-F TGCATCATCATCATCATCATgctggcagggaagcagatg P4HB-pCOLDII recombinant-R TTTTAAGCAGAGATTACCTAgccctccaggaatttattacagaa

[0046] (2) Expression and purification of recombinant proteins:

[0047] Using chicken cDNA as template, P4HB-pCOLDII recombinant-F, P4HB-pCOLDII recombinant-R as primers, the cDNA fragment corresponding to P4HB was amplified by PCR method. The PCR amplification system was 20μL: PrimeSTARMax Premix (2×) 10μL, 1μL of upstream and downstream primers, 1μL of DNA template, and ddH2O was added to 20μL. PCR reaction conditions: 95℃ pre-denaturation for 1min; 95℃ denaturation for 15s, 60℃ annealing for 30s, 72℃ extension for 30s, a total of 35 cycles; 72℃ extension for 10min; at the same time, pCOLDII empty vector was linearized using linear primers. After the PCR product and linearized product were identified by agarose gel electrophoresis, they were recovered with a PCR product purification kit, and then the two products were connected by homologous recombinase. The ligation product was transformed into competent TOP10 and coated on an ampicillin-resistant LB plate, and incubated overnight at 37℃ for 12h. After the bacterial solution was correctly identified by PCR, it was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0048] The recombinant plasmid with correct sequencing was extracted according to the instructions of the small extraction plasmid kit, named pCOLDII-P4HB, and stored at -20℃. The recombinant plasmid pCOLDII-P4HB was transformed into E. coli BL21 (DE3) competent cells, cultured at 37℃ and 220r / min until the OD600nm value was 0.4-0.6, and IPTG with a final concentration of 0.5mol·L-1 was added, and induced at 16℃ and 220r / min for 20h. The bacteria were collected, ultrasonically disrupted and centrifuged, and the supernatant and precipitate were collected and the expression of the recombinant protein was detected by SDS-PAGE ( Figure 4 ). Purification was performed by Ni-NTA affinity chromatography. The flow-through, washing solution and eluate in the purification process were prepared and run on SDS-PAGE gel. The results showed that the target protein was in the eluate and its size was 25KDa ( Figure 5 ). Then, purified HIS-P4HB recombinant protein was obtained through large-scale prokaryotic expression and purification. The concentration of purified protein was determined by BCA method and then stored at -80℃.

[0049] (3) Preparation of polyclonal antibodies:

[0050] After the purified HIS-P4HB recombinant protein was mixed and emulsified with an equal volume of Freund's complete adjuvant, multiple subcutaneous injections were performed at the back of the neck, with an immunization dose of 200 μg / mouse. The second and third immunizations were performed at the third and fifth weeks after the first immunization, respectively, both of which were emulsified with Freund's incomplete adjuvant, and the method and dose were the same as the first immunization. On the 9th day after the third immunization, blood was collected from the ear vein to separate serum. The fourth immunization was performed at the 7th week after the first immunization. One week later, blood was collected from the heart, serum was separated, diluted with an equal volume of PBS, and filtered with a 0.22 μM filter. Protein A+G was used to bind overnight at 4°C and then eluted and purified with 0.1M glycine (PH3.0) eluent. After dialysis in PBS, 50% glycerol was added and stored at -80°C.

[0051] (4) Determination of anti-chicken P4HB polyclonal antibody titer:

[0052] The His-P4HB protein was diluted to 0.6 μg / mL with the coating solution, coated on a 96-well ELISA plate, 100 μL / well, and incubated at 4°C overnight; washed 3 times with PBST; added blocking solution (5% skim milk solution), 150 μL / well, and blocked at 37°C for 2 hours; washed in the same manner as above; the above-concentrated anti-chicken P4HB polyclonal antibody was diluted in multiples from 1:200 with PBS, 100 μL / well, and incubated at 37°C for 1 hour; washed in the same manner as above; HRP-labeled goat anti-mouse IgG was diluted to a working concentration (1:10000) with 5% skim milk, 100 μL / well, and incubated at 37°C for 1 hour; washed in the same manner as above; added 100 μL of TMB to each well, and developed the color for 15 minutes in the dark; added 50 μL of 2MH2SO4 to each well to terminate the reaction, and the OD450 value of each well was measured by an ELISA instrument. When the polyclonal antibody serum OD / negative control serum OD>2.1, the maximum dilution multiple among the positive samples is the serum titer.

[0053] (5) Western Blot verification of the specificity of the rabbit anti-chicken P4HB polyclonal antibody of the present invention:

[0054] The prepared His-P4HB protein was subjected to SDS-PAGE electrophoresis according to the conventional method; the protein on the PAGE gel was transferred to a PVDF membrane; the PVDF membrane was blocked with 5% skim milk at room temperature for 3 hours; the anti-chicken P4HB polyclonal antibody was diluted 1:1000 with 5% skim milk as the primary antibody, incubated at 4°C overnight, and washed with TBST 5 times, each time for 5 minutes; HRP-labeled goat anti-rabbit IgG antibody was diluted 1:10000 as the secondary antibody, incubated at room temperature for 1 hour; after washing, the color was developed and exposed.

[0055] (6) Evaluation of the application of the rabbit anti-chicken P4HB polyclonal antibody of the present invention in Western Blot

[0056] We selected the representative chicken cell line HD11 and chicken liver tissue, extracted proteins, and performed Western Blot application evaluation to detect the expression of proteins after linearization of native proteins.

[0057] Chicken HD11 cell line protein treatment: HD11 was plated in a 12cm dish, cultured overnight, cells were collected, cells were lysed with cell lysis buffer, and proteins were collected. Chicken liver tissue protein treatment: A small amount of chicken liver was placed in a 2ml cryo-grinder special grinding tube, one large steel ball and two small steel balls were added, 400μL of single detergent lysis buffer (containing PMSF) was added, and the frequency of the cryo-grinder was set to 65Hz, and the grinding was performed for 2min. After the grinding, the lysate can be transferred to a 1.5ml centrifuge tube with a pipette, placed on ice for 30min, and then centrifuged at 12000rpm for 5min at 4℃, and the supernatant was added with protein loading buffer and boiled for loading.

[0058] The specific operation of Western Blot is as follows: perform SDS-PAGE according to the conventional method; transfer the protein on the PAGE gel to the PVDF membrane; block the PVDF membrane with 5% skim milk at room temperature for 3 hours; dilute the above-mentioned anti-chicken P4HB polyclonal antibody with 5% skim milk at 1:1000 as the primary antibody, incubate at 4°C overnight, wash with TBST 5 times, 5 minutes each time; use HRP-labeled goat anti-rabbit IgG antibody as the secondary antibody after diluting it at 1:10000, incubate at room temperature for 1 hour; after washing, color development and exposure.

[0059] (7) Evaluation of the application of the rabbit anti-chicken P4HB polyclonal antibody of the present invention in IF experiments

[0060] In the IF experiment, the intracellular protein maintains its native conformation. Through this experiment, we can evaluate the ability of the rabbit anti-chicken P4HB polyclonal antibody of the present invention to detect the expression and cellular localization of the chicken P4HB protein in the native conformation.

[0061] When HD11 cells grow to 50% in a 12cm dish, discard the culture medium, add trypsin for 2-3 minutes, terminate with DMEM, and centrifuge at 1000rpm for 3 minutes. Discard the supernatant, add DMEM to 10ml, spread on a 24-well plate, and culture at 37 degrees overnight. Use IF method to detect the specific reaction of P4HB antibody with endogenous P4HB protein, and detect the expression level and cell localization of endogenous P4HB protein. The specific operation is as follows: discard the cell solution, wash with PBS, add 200 μL of 4% cell tissue fixative to each well, and place at room temperature for 20 minutes; discard the fixative, wash with PPS, add 150 μL of 0.1% TRITON-100 to each well, and place at 37°C for 15 minutes; discard the transmembrane solution, wash with PBS, add 200 μL of 5% BSA to each well, and place at 37°C for 1 hour; discard the blocking solution, wash with PBS, dilute the above-mentioned anti-chicken P4HB polyclonal antibody with PBS at 1:100 times as the primary antibody, add 100 μL to each well, and incubate at 37°C for 2 hours; after washing with PBS, use 488 fluorescently labeled goat anti-rabbit IgG antibody at a dilution of 1:10000 as the secondary antibody, and incubate at room temperature for 1 hour; after washing with PBS, add 50 μL of DAPI staining solution to each well, stain for 10 minutes, and observe using an inverted microscope.

[0062] Example 2

[0063] (1) Expression and purification of P4HB protein:

[0064] Using chicken cDNA as template, the P4HB fragment amplified by PCR was consistent with the predicted size (732 bp). Figure 2 The obtained chicken P4HB fragment was recombined into the pCOLDII prokaryotic expression vector to obtain a recombinant expression plasmid. The bacterial liquid PCR identification showed that the gene fragment size of the P4HB-2 strain was consistent with ( Figure 3 ). The results of gene sequencing showed that the P4HB-2 recombinant plasmid pCOLDII-P4HB was constructed correctly. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced for expression. The SDS-PAGE electrophoresis results showed a clear band at 25KDa, and the protein was expressed in the cell supernatant ( Figure 4 ). The protein purity is high after purification ( Figure 5 ). The above results show that we have obtained P4HB recombinant protein with high purity. The protein concentration determined by BCA method is 4 mg·mL-1, and its absorbance after dilution 10 times is 0.426.

[0065] (2) Purification of chicken P4HB polyclonal antibody:

[0066] After the rabbits were immunized with P4HB recombinant protein 4 times, blood was collected and serum was separated. The serum was then bound using a ProteinA+G purification column at 4°C overnight and then eluted and purified using a 0.1M glycine (PH3.0) eluent. The purification process curve is shown in Figure 6 As shown. Blue represents the UV absorption value. The first UV peak is the impurity protein flowing through the antiserum; the second UV peak is the eluted IgG that can specifically bind to the ligand, that is, the polyclonal antibody. Red represents the ionic strength value. Displays the ionic strength values ​​of different solutions flowing through the column

[0067] (3) Determination of the titer of anti-chicken P4HB polyclonal antibody

[0068] The His-P4HB protein was diluted to 0.6 μg / mL with the coating solution, and coated on a 96-well ELISA plate at 100 μL / well. The plate was incubated at 4°C overnight. After blocking with skim milk, the anti-chicken P4HB polyclonal antibody was diluted from 1:200 and incubated at 37°C for 1 hour. Then, the HRP-labeled goat anti-mouse IgG was diluted to the working concentration (1:10000) and incubated at 37°C for 1 hour. After the TMB color development solution was added and the plate was protected from light for 15 minutes, 50 μL 2M H2SO4 was added to each well to terminate the reaction. The OD450 value of each well was measured by an ELISA instrument. The measured values ​​(Table 2) and the change curve ( Figure 7 ), the titer of the serum before purification was 204800, and the titer of the purified anti-chicken P4HB polyclonal antibody was 102400.

[0069] Table 2

[0070]

[0071] (4) Western Blot verification of the specificity of the rabbit anti-chicken P4HB polyclonal antibody of the present invention

[0072] The prokaryotic expressed HIS-P4HB recombinant protein was prepared, and the loading amount gradient was set, i.e., 5 μg; 1 μg; 0.5 μg; 0.1 μg. The Western blot results showed that the rabbit anti-chicken P4HB polyclonal antibody of the present invention could react with the recombinant protein to produce a specific band of nearly 25 KDa, and the gray value was dose-dependent with the sample content, indicating that the rabbit anti-chicken P4HB polyclonal antibody of the present invention had good specificity ( Figure 8 ).

[0073] (5) Evaluation of the application of the rabbit anti-chicken P4HB polyclonal antibody of the present invention in IF experiments

[0074] Using the rabbit anti-chicken P4HB polyclonal antibody of the present invention as the primary antibody, IF results showed that specific green fluorescence appeared in HD11, located in the cell membrane and cytoplasm ( Fig.10). These results indicate that the rabbit anti-chicken P4HB polyclonal antibody of the present invention can be used to detect the expression and cellular localization of the chicken P4HB antigen in its native conformation.

[0075] (6) Evaluation of the application of the rabbit anti-chicken P4HB polyclonal antibody of the present invention in Western Blot The results of Western blot detection showed that the rabbit anti-chicken P4HB polyclonal antibody of the present invention can bind to HD11 ( Fig. 9 ) and chicken liver tissue ( Fig.11 ) to produce a specific band of approximately 55 KDa.

[0076] The structure of the antigenic determinant can be caused by a variety of reasons, including the specific sequence of the antigen molecule, the spatial arrangement of amino acid residues, chemical modification, etc. The chicken P4HB natural protein contained in serum, cell lines and tissues has a complete natural conformation, presents a unique three-dimensional spatial configuration and exposes specific unknown antigen sequences. These unique spatial sequences and specific sequences constitute the antigenic determinant of the chicken P4HB natural protein. Prokaryotic expression has the characteristics of fast expression speed, low cost and high protein expression, but the antigens synthesized by different antigen sequences and prokaryotic expression methods may be different from the proteins of natural conformation. The present invention selects the 123aa-354aa polypeptide of the chicken P4HB protein as an antigen, constructs a prokaryotic expression vector, and performs prokaryotic expression under specific conditions to obtain a soluble antigen polypeptide as an immunized antigen. Compared with the precipitated antigen in the inclusion body, the soluble antigen has the advantage of being closer to the spatial structure of the natural protein.

[0077] The present invention uses prokaryotically expressed chicken P4HB protein to immunize New Zealand white rabbits and successfully prepares specific rabbit anti-chicken P4HB polyclonal antibodies. IF experimental results show that the antibody can effectively recognize the chicken P4HB protein in its native conformation and specifically bind to it, and is used for detecting the expression and cellular localization of the chicken P4HB protein in its native conformation.

[0078] In the Western blot experiment, the protein was boiled in a buffer containing SDS and DTT, and its natural conformation was destroyed, existing in a straight chain form, forming a linearized chicken P4HB protein, whose antigenic determinant cluster has undergone a huge change. The results of Western blot detection of chicken P4HB protein in chicken tissues and HD11 cell lines showed that although the antigenic determinant cluster of the natural chicken P4HB protein has undergone a huge change, the antibody prepared by our invention can still recognize and bind well, and has a very high titer.

[0079] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

[0080] The sequence of chicken P4HB polypeptide is SEQ ID NO.1:

[0081] MAVVRVRAIVALLCLVAALGLAEPLEEEDGVLVLRAANFEQALAAHRHLLVEFYAPWCGHCKALAPEYAKAAAQLKAEGSEIRLAKVDATEEAELAQQFGVRGYPTIKFFRNGDKAAPREYTAGREAD DIVSWLKKRTGPAATTLTDAAAAETLVDSSEVVVIGFFKDVTSDAAKEFLLAAESVDDIPFGISSSADVFSKYQLSQDGVVLFKKFDEGRNNFEGDLTKDNLLNFIKSNQLPLVIEFTEQTAPKIFGGE IKTHILLFLPKSVSDYEGKLDNFKTAAGNFKGKILFIFIDSDHSDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESDDLTADKIKEFCNKFLEGKIKPHLMSQDLPEDWDKQPVKVLVGKNFEEVA FDENKNVFVEFYAPWCGHCKQLAPIWDKLGETYRDHENIVIAKMDSTANEVEAVKIHSFPTLKFFPAGSGRNVIDYNGERTLEGFKKFLESGGQDGAAADDDLEDLETDEETDLEEGDDDEQKIQKDEL

[0082] The amino acid sequence selected is SEQ ID NO.2:

[0083] AGREADDIVSWLKKRTGPAATTLTDAAAAETLVDSSEVVVIGFFKDVTSDAAKEFLLAAESVDDIPFGISSSADVFSKYQLSQDGVVLFKKFDEGRNNFEGDLTKDNLLNFIKSNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYEGKLDNFKTAAGNFKGKILFIFIDSDHSDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESDDLTADKIKEFCNKFLEG。

[0084] Amino acid sequence of the chicken HIS-P4HB modified polypeptide SEQ ID NO.3:

[0085] MNHKVHHHHHHAGREADDIVSWLKKRTGPAATTLTDAAAAETLVDSSEVVVIGFFKDVTSDAAKEFLLAAESVDDIPFGISSSADVFSKYQLSQDGVVLFKKFDEGRNNFEGDLTKDNLLNFIKSNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYEGKLDNFKTAAGNFKGKILFIFIDSDHSDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESDDLTADKIKEFCNKFLEG

Claims

1. A chicken HIS-P4HB modified polypeptide, characterized in that: The amino acid sequence of the chicken HIS-P4HB modified polypeptide is shown in SEQ ID NO.

3.

2. The chicken HIS-P4HB modified polypeptide according to claim 1, characterized in that: The chicken HIS-P4HB modified polypeptide is obtained by screening a peptide segment of the chicken P4HB polypeptide shown in SEQ ID NO.2, performing histidine tag modification, and then expressing it in prokaryotic cells.

3. A polyclonal antibody against chicken HIS-P4HB modified polypeptide, characterized in that: The polyclonal antibody of the chicken HIS-P4HB modified polypeptide is obtained by immunizing the chicken HIS-P4HB modified polypeptide according to claim 1 as an antigen.

4. The method for preparing a polyclonal antibody against a chicken HIS-P4HB modified polypeptide according to claim 3, characterized in that: include: The chicken P4HB gene was amplified by PCR and homologously recombined into the expression vector to construct the prokaryotic recombinant expression plasmid pCOLDII-P4HB; The recombinant plasmid was transformed into E.coli BL21 and induced for expression with IPTG. The protein was then purified using Ni-NTA affinity chromatography. The purified HIS-P4HB protein was mixed with an equal volume of Freund's complete adjuvant for emulsification and then immunized and the serum was separated. The polyclonal antibody was obtained after purification by Protein A + G affinity chromatography.

5. The preparation method according to claim 4, characterized in that: The immunization conditions are to use multiple subcutaneous injections at the back of the neck, and the second and third immunizations are respectively performed at the 3rd and 5th weeks after the first immunization, both of which are emulsified with Freund's incomplete adjuvant, and the method and dosage are the same as the first immunization.

6. The preparation method according to claim 5, characterized in that: The immunization dose was 200 μg / mouse.

7. The preparation method according to claim 5, characterized in that: On the 9th day after the three immunizations, blood was collected from the ear vein to separate serum; the fourth immunization was performed on the 7th week after the first immunization, and blood was collected from the heart one week later.

8. The preparation method according to claim 4, characterized in that: The prokaryotic recombinantly expressed HIS-P4HB protein is expressed in the supernatant of E.coli BL21 cells, and has a molecular weight of about 25 KDa.

Images