CN112759645A

CN112759645A - Chicken RPS14 polyclonal antibody and preparation method and application thereof

Info

Publication number: CN112759645A
Application number: CN202110202465.5A
Authority: CN
Inventors: 刘平; 胡国良; 郭小权; 顾玥明; 施燕; 刘佩; 曹华斌; 程素芳; 李麟; 李谷月; 黄橙; 赵玉兰; 郭凤萍; 李庆庆; 方玮乐
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2021-02-23
Filing date: 2021-02-23
Publication date: 2021-05-07

Abstract

The invention discloses a chicken RPS14 polyclonal antibody and a preparation method and application thereof, belonging to the technical field of biology; the invention utilizes specific primers to combine with corresponding templates to carry out PCR amplification of RPS14 genes, and then utilizes TA cloning and subcloning technologies to construct recombinant expression plasmid PET28a-RPS 14; purifying and dialyzing the recombinant RPS14 protein induced and expressed by isopropyl-beta-D-thiogalactoside (IPTG) to immunize rabbits as an antigen, thereby preparing high-quality polyclonal antibodies; the preparation method of the polyclonal antibody is simple and convenient to operate and low in cost, the prepared polyclonal antibody can be used for detecting RPS14 protein in animal organisms, a material foundation is laid for research and diagnosis of pathological mechanisms of various broiler diseases, and the preparation method has the advantages of wide application range and strong specificity.

Description

Chicken RPS14 polyclonal antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a chicken RPS14 polyclonal antibody and a preparation method and application thereof.

Background

Broiler Ascites Syndrome (AS), also known AS pulmonary arterial hypertension syndrome (PHS), is a non-infectious nutritional metabolic disease that occurs primarily in broiler bodies. Its clinical features are thin feathers, abnormal gait, listlessness, poor appetite accompanied by marked abdominal distension. Research shows that the diseases are mostly caused by genetic, nutritional, environmental and feeding management factors and are high in the fast-growing broiler chicken group. The reason is that the muscle mass and the metabolic rate of the fast growing broiler chickens are obviously improved, and the consumption of oxygen is also obviously increased. However, the volume and volume of the heart and lung are not increased, so the cardiopulmonary system cannot meet the increasing oxygen demand, and further the cardio-pulmonary artery vasoconstriction is caused, and the tissue hypoxia is serious. Hypoxia in turn causes increased arterial blood pressure, right ventricular hypertrophy with rapid failure. Research has shown that the pulmonary hypertension and ascites diseases cause serious harm to the commercial broiler breeding industry in China, the broiler diseases are easy to occur in cold winter and early spring, and are accompanied by obvious mass-sending property, the fatality rate is 10% -30%, and the disease has the trend of rising year by year. In the broiler industry worldwide, ascites diseases cause about 25% of total mortality, a disease that seriously impairs the economic benefits of the poultry industry in recent years.

The RPS14(ribosomal protein S14) gene is a member of the ribosomal protein gene superfamily. The gene is located at the 3' end of 18S rRNA and consists of 5 exons and 4 introns, and plays a key auxiliary role in the formation process of a ribosome 40S subunit complex. Research shows that the expression of RPS14 gene and excessive apoptosis of red blood cells show obvious negative correlation trend. When the animal body is subjected to a series of physiological stress reactions, such as: ribosome deficiency stabilizes and activates the p53 gene by inhibiting MDM 2-mediated polyubiquitination (degradation) of p53, and activation of the p53 pathway leads to arrest of the growth cycle of erythrocytes and promotes their massive apoptosis. The rapid reduction of erythrocytes can cause the obvious shortage of oxygen supply in the heart and lung of animals, and when the oxygen supply capacity of cells and tissues is not matched with higher metabolic load, the body has obvious hypoxia to induce the generation of ascites syndrome. Meanwhile, the RPS14 gene also has the ability of influencing the normal biosynthesis of ribosome and changing the translation of protein, the activation and expression of ribosomal protein S14 are the premise of globin synthesis, and globin is the basis of erythrocyte synthesis, thereby influencing the production of hemoglobin. Hemoglobin is a special protein for transporting oxygen in erythrocytes, and the lack of the protein can cause insufficient oxygen transportation of heart and lung, so that obvious tissue hypoxia occurs, which can cause the increase of vascular resistance and pressure enhancement, and finally causes the occurrence of broiler ascites syndrome. In conclusion, RPS14 has a close relationship with diseases such as pulmonary hypertension and ascites of broiler chicken. In the earlier studies of the present invention, it was demonstrated that the expression of RPS14 gene mRNA was reduced in broiler chickens suffering from such diseases as compared to the normal group. This indicates that at the molecular level, expression of RPS14 has a significant impact on the development of this disease. However, the effect on such diseases at the protein level has not been clear. Meanwhile, the detection and pathological mechanism research of ascites diseases of broiler chickens by preparing RPS14 polyclonal antibodies are hardly reported.

Disclosure of Invention

The invention aims to provide a chicken RPS14 polyclonal antibody, a preparation method and application thereof, so as to solve the problems in the prior art.

In order to achieve the aim, the invention provides a preparation method of a chicken RPS14 gene polyclonal antibody, which comprises the following steps: (1) taking chicken cDNA as a template, carrying out PCR amplification of RPS14 gene to obtain a target fragment, wherein the nucleotide sequence of the primer amplified by the PCR is shown as SEQ ID NO: 1-2; (2) connecting the target fragment with a PET-28a (+) expression vector to construct a recombinant expression plasmid PET28a-RPS 14; (3) transferring the recombinant expression plasmid PET28a-RPS14 into a Rosetta (DE3) competent cell, performing prokaryotic expression, purifying, and performing dialysis renaturation to obtain chicken RPS14 recombinant protein; (4) taking blood after immunizing rabbits with the chicken RPS14 recombinant protein, and separating serum to obtain the chicken RPS14 gene polyclonal antibody; (5) and performing antibody quality verification on the chicken RPS14 gene polyclonal antibody by using enzyme-linked immunosorbent assay, protein immunoblotting detection or immunofluorescence technology.

Further, the PCR amplification procedure of step (1) is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 35s, and extension at 72 ℃ for 40s for 35 cycles; extension at 72 ℃ for 10 min.

Further, the prokaryotic expression in the step (3) comprises the following specific steps: to Rosetta (DE3) competent cells, which were transferred to PET28a-RPS14, IPTG was added to a final concentration of 1mM and shaken for 4h at 37 ℃ on a shaker at 220 rpm/min.

Further, the eluent purified in the step (3) is an eluent with the concentration of 500mM imidazole.

Further, the dialysis renaturation in the step (3) is to dialyze the target protein by using urea gradient descent, and the dialyzate is replaced every 4 hours in an environment at 4 ℃.

Further, the specific method for immunizing the white rabbit with the chicken RPS14 recombinant protein in the step (4) comprises the following steps: the chicken RPS14 recombinant protein was injected subcutaneously in New Zealand white rabbits at multiple sites to elicit an immune response, which was boosted every 10 days for a total of 30 days.

Further, the optimal dilution concentration of the antigen coating in the enzyme-linked immunosorbent assay in the step (5) is 2.5 mu g/ml; the dilution gradient of both the anti-RPS 14 serum and the pre-immune serum was 1:100 to 1: 204800; the dilution concentration of goat anti-rabbit IgG labeled by horseradish peroxidase is 1: 3000A; the reaction wavelength of the microplate reader is 450 nm;

the dilution ratio of the anti-RPS 14 serum to the pre-immune serum in the protein immunoblot detection is 1: 3000A; the dilution ratio of goat anti-rabbit IgG labeled by horseradish peroxidase is 1: 1000, parts by weight;

the dilution ratio of the anti-RPS 14 serum to the pre-immune serum in the immunofluorescence technique is 1: 200 of a carrier; the dilution ratio of CY3 labeled goat anti-rabbit IgG (H + L) was 1: 300.

the invention also provides a chicken RPS14 polyclonal antibody prepared by any one of the preparation methods.

The invention also provides application of the chicken RPS14 polyclonal antibody in preparation of a tool for detecting chicken pulmonary hypertension and/or ascites diseases.

Further, the means is a reagent or a kit.

The invention discloses the following technical effects: the invention adopts cloning and subcloning technology to construct a PET28a-RPS14 expression vector, and the recombinant RPS14 protein expressed by isopropyl-beta-D-thiogalactoside (IPTG) induction is purified and then used as an antigen to immunize rabbits, thereby preparing high-quality polyclonal antibodies. The detection method of enzyme-linked immunosorbent assay, protein immunoblotting, indirect immunofluorescence technology and the like is utilized to detect the RPS14 gene expression so as to verify the sensitivity and specificity of the polyclonal antibody, and the chicken RPS14 polyclonal antibody prepared by the invention has the advantages of good sensitivity, strong specificity, wide application range and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a technical scheme diagram of a development method of an RPS14 gene polyclonal antibody related to the occurrence and development of broiler pulmonary hypertension and ascites diseases;

FIG. 2 shows the construction of cloning and expression recombinant plasmids; wherein, A is RCR amplification product (453bp) of RPS14 gene, lanes 1-2 are PCR amplification bands of target gene, and lane 3 is negative control; b is

-PCR result of T3-RPS14 plasmid, lane 1-2 shows the RPS14 gene fragment inserted into the recombinant clone plasmid, and lane 3 shows the negative control; c is a PCR result chart of the bacterial liquid of the PET28a-RPS14 plasmid, lane 1 is a negative control, and lanes 2-3 are target fragments inserted in the expression recombinant plasmid; d is a recombinant plasmid double-restriction enzyme identification result diagram, lane 1 is a negative control, and lane 2 is double-restriction enzyme-digested recombinant PET28a-RPS 14; in the figure, M is a standard DNA marker (2000/5000).

FIG. 3 shows prokaryotic expression and purification of recombinant RPS14 protein, wherein A is successfully expressed recombinant RPS14 protein, lanes 1-2 are total protein expressed by prokaryotic RPS14 gene, and lane 3 is blank control (without inducer); b is the identification of the expression form of recombinant RPS14 protein, lane 1 is the supernatant after ultrasonication, lane 2 is the pellet after ultrasonication, lane 3 is a blank control (no inducer added); c is the recombinant RPS14 protein inclusion body dissolution identification, lane 1 is the supernatant after ultrasonic disruption, lane 2 is the precipitate after ultrasonic disruption, lane 3 is the supernatant after the RPS14 inclusion body dissolution; d is RPS14 protein after nickel column purification, lanes 1-3 are RPS14 protein (22kDa) after purification indicated by white arrows, lane 4 is elution buffer; e is the dialyzed concentrated recombinant protein, lanes 1-4 are the concentrated protein of interest (22kDa), the standard protein Marker size of the five electropherograms is 10-180kDa, and the white arrows indicate the protein of interest.

FIG. 4 shows the measurement of the titer of anti-RPS 14 serum, wherein the X-axis represents the dilution gradient of anti-RPS 14 serum, the Y-axis represents the value of anti-RPS 14 serum at 450nm, antiserum 1-3 represents the absorbance of the antiserum from three different rabbits at the same dilution gradient, and the preimmune serum is the absorbance of the nonimmune serum at the same dilution gradient.

FIG. 5 is a method for detecting the expression level of RPS14 protein in white feather broilers by an immunoblotting method; a is the protein level of RPS14 in five related tissues; b is an expression histogram of RPS14 protein in a normal group and a pathological group, wherein the X axis is five different tissues of pulmonary artery, liver, kidney, heart and lung, the Y axis is the expression level of RPS14 compared with beta-actin, and statistically analyzed data are expressed in a mean +/-SE mode (n is 3); c is the specificity of RPS14 protein in prokaryotic expression, the upper lane 1-3 is the broiler pulmonary artery RPS14 protein (the black arrow indicates the position is 22kDa), and the lower lane 1-3 is a negative control (primary antibody: preimmune serum).

FIG. 6 is a graph of immunofluorescence results of protein expression immunofluorescence assay of meat chicken, meat duck, goat, mouse and rabbit related tissues (pulmonary artery, liver, kidney, heart and lung) RPS14, wherein A-F are PBS buffer (blank control), preimmune serum (negative control), rabbit anti-chicken RPS14 serum, normal meat chicken, ascites meat chicken, normal meat duck, normal goat, normal mouse and normal rabbit related tissues after section incubation; g is a histogram of the average fluorescence intensity of the RPS14 protein in the pulmonary artery, liver, kidney, heart and lung tissues of the broiler chicken, wherein the X axis is related tissues, and the Y axis is the average fluorescence intensity of the RPS14 protein; h is a graph of the results of homology comparison of RPS14 genes of different species; i is a histogram of the mean fluorescence intensity of RPS14 protein in related tissues of different animals, wherein the X axis is the pulmonary artery, liver, kidney, heart and lung, and the Y axis is the mean fluorescence intensity of RPS14 protein.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

Unless otherwise specified, the reagents used in the following examples are commercially available.

EXAMPLE 1 broiler RPS14 Gene polyclonal antibody preparation

1. Construction of PET28a-RPS14 recombinant expression vector

1.1 according to the nucleotide sequence of chicken RPS14 gene reported in Genbank, screening high-quality nucleotide coding sequence by bioinformatics analysis, designing and synthesizing specific primer, and carrying out corresponding combination with template to amplify RPS14 gene.

The specific primers are as follows:

F：5’-CGCGGATCCATGGCACCCCGCAAG-3’(SEQ ID NO：1)；

R：5’-CCCAAGCTTCAGACGACGTCCACG-3’(SEQ ID NO：2)。

the 5 'end of the upstream primer of the specific primer is inserted with BamHI restriction endonuclease, and the 5' end of the downstream primer is inserted with HindIII restriction endonuclease.

The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 35s, and extension at 72 ℃ for 40s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.

1.2 inserting the target fragment into the plasmid by TA cloning

-T3 vector, thereby obtaining recombination

-T3-RPS14 cloning vector.

1.3 pairs of the recombinant cloning vectors

Carrying out double digestion on the T3-RPS14 and an empty expression vector PET-28a (+), connecting the obtained target fragment with the PET-28a (+) expression vector, and constructing a PET28a-RPS14 recombinant expression plasmid.

1.4 transferring the recombinant expression plasmid PET28a-RPS14 into a Rosetta (DE3) competent cell to obtain a transformed competent cell.

2. Acquisition of anti-RPS 14 protein

2.1 adding IPTG to the transformed competent cells to a final concentration of 1mM, shaking the cells on a shaker at 37 ℃ and 220rpm/min for 4 hours, and subjecting the obtained protein to 15% SDS-PAGE to determine whether the protein is the desired target protein. As can be seen from fig. 3A, recombinant RPS14 protein was abundantly expressed under these conditions and indicated by white arrows.

2.2 the RPS14 protein after induction expression is identified in expression form, the supernatant and the precipitate are separated by ultrasonic disruption and centrifugation, as shown in FIGS. 3B-C, the recombinant protein is stored in the precipitate in a large amount in the form of inclusion bodies, and soluble protein with corresponding size (22kDa) can be obtained from the supernatant after dissolution.

2.3 the dissolved RPS14 protein is purified by nickel affinity column, elution purification is carried out by using elution buffer solution with 500mM imidazole concentration, identification and analysis are carried out by 15% SDS-PAGE electrophoresis, the purified protein is stored for dialysis and renaturation, as shown in figure 3D, elution buffer solution with 500mM imidazole concentration is used for eluting protein, and the RPS14 recombinant protein is purified by nickel affinity column to obtain the target protein with 22kDa molecular weight and single definition, which is consistent with the expected size.

2.4 carrying out dialysis renaturation on the purified protein, dialyzing the target protein by adopting the gradient descending trend of urea, and replacing the dialysate every 4 hours in an environment at 4 ℃; then concentrated by PEG8000 and used for rabbit immunization. As shown in FIG. 3E, the target band after electrophoresis is clearer and more uniform and has a darker color, which indicates that the purity and concentration of the protein are improved after concentration by dialysis.

3. Preparation of RPS14 polyclonal antibody

Mixing the RPS14 protein subjected to dialysis and renaturation with Freund's complete adjuvant in equal volume, performing subcutaneous multi-point injection on the rabbit, performing boosting immunization once every 10 days (the boosting immunization requires that the RPS14 protein is mixed with Freund's incomplete adjuvant), taking the ear artery blood after 30 days of immunization, and centrifuging to obtain immune serum, thereby obtaining the RPS14 polyclonal antibody.

Example 2 quality validation of RPS14 polyclonal antibody

2.1 enzyme-linked immunosorbent assay

The purified recombinant RPS14 protein was diluted to 2.5. mu.g/ml as an antigen, plated on 96-well plates (150. mu.L/well) and incubated at 37 ℃ for 6 hours. After 3 washes with PBS-T, 200. mu.l of 5% nonfat dry milk blocking solution was added to each well, incubated at 37 ℃ for 2 hours, washed, serially diluted (1:100-1:204800) anti-RPS 14 serum and pre-immune serum (100. mu.L/well) with the same dilution gradient were added, and after 1h of incubation at 37 ℃ and washed, the mixture was added according to the following formula 1: 3000 diluted goat anti-rabbit IgG (120 mu L/well) marked by horseradish peroxidase is incubated for 1 hour, and finally, PBS-T is used for thorough cleaning, then soluble single-component TMB substrate solution is added for reaction in a dark place for 20min, concentrated sulfuric acid is used for stopping the reaction, and the absorbance value of 450nm is taken in an enzyme-linked immunosorbent assay for measurement, thereby calculating the actual titer of the RPS14 polyclonal antibody.

As can be seen from FIG. 4, the previous experiments show that the optimal antibody titer can be obtained by selecting the antigen coating concentration of 2.5. mu.g/ml, and the serum titer of the rabbit anti-chicken RPS14 extracted from 3 different New Zealand white rabbits is as high as 1 according to the calculation formula that OD positive/OD negative is not less than 2.1: 204,800.

2.2 Western Blot detection

After extracting total proteins of tissues of broilers in a normal group and a pathological group, performing 15% SDS polyacrylamide gel electrophoresis on the total proteins, taking a stably expressed gene beta-actin as an internal reference, transferring an electrophoresis strip onto a polyvinylidene fluoride membrane (PVDF) through wet transfer, sealing the PVDF membrane in 5% skimmed milk powder for 2 hours at room temperature, then incubating the PVDF membrane on a shaking table at 4 ℃ overnight by using anti-RPS 14 serum (diluted by 1: 3000), washing the PVDF membrane for 3 times (washing for 10min each time) by using TBS-T, incubating the membrane and goat anti-rabbit IgG (diluted by 1: 1000) marked by horseradish peroxidase for 1 hour at room temperature, finally washing the PVDF membrane for 3 times, performing protein strip color development by using a high-sensitivity ECL chemiluminescence detection kit, and further detecting the protein level of the gene in the tissues.

As can be seen from FIGS. 5A-B, rabbit anti-chicken RPS14 positive serum as a primary antibody can specifically bind to total protein extracted from five tissues (pulmonary artery, liver, kidney, heart and lung) of broiler chicken. And the statistical method is used for analysis, so that the expression level of the RPS14 protein in the ascites group in the five tissues shows a descending trend, and the expression level of the RPS14 protein in pulmonary artery, liver, heart and lung of the ascites chicken is lower than that in a control group (p is less than or equal to 0.01) by a very significant difference.

Meanwhile, rabbit anti-chicken RPS14 positive serum and pre-immune serum are used as primary antibodies to carry out specificity detection on the RPS14 protein expressed by pronucleus, as shown in a result of figure 5C, the pre-immune serum cannot be combined with the RPS14 protein expressed by pronucleus, and a PVDF membrane incubated by the rabbit anti-chicken RPS14 positive serum has obvious protein expression, so that the polyclonal antibody has obvious reactogenicity on the RPS14 protein.

2.3 Indirect immunofluorescence technique

After antigen retrieval, deparaffinized slides about 5 μm thick were blocked in goat serum for 30 minutes. The slides were incubated overnight at 4 ℃ with Anti-RPS 14 protein antibody (1: 200 dilution) and preimmune serum (1: 200 dilution), washed 3 times (5 min each) with PBS-T, and incubated with Cy3 conjugated Goat Anti-Rabbit IgG (H + L) incubation (Boster, Wuhan, China) diluted 1:300 for 50min at room temperature in the absence of light. Washing with PBS-T for 3 times (5 min each time), dripping 4',6-diamidino-2-phenylindole (DAPI; 1:400dilution), incubating in dark for 10min, washing for 3 times, sealing with fluorescence quenching sealing agent, and positioning under Nikon inverted fluorescence microscope.

As shown in fig. 6A-F, the PBS buffer, preimmune serum and rabbit anti-chicken RPS14 serum were used as primary antibodies to incubate with the relevant tissues (pulmonary artery, liver, kidney, heart and lung) of broiler chicken, meat duck, goat, mouse and rabbit, and it was found that the experimental group showed bright fluorescence signal and had strong positive compared to the two negative controls, indicating that the polyclonal antibody can specifically bind to RPS14 protein in animal tissues, and that the expression of RPS14 protein was mainly concentrated in the cytoplasmic portion of various organs of animals. As seen in FIG. 6G, statistical analysis of fluorescence intensity of the broiler normal group and ascites group showed that the expression level of RPS14 protein in the broiler related tissues was significantly higher in the normal group than in the ascites group (p.ltoreq.0.01). As shown in FIG. 6H, I, the homology comparison of the broiler chicken RPS14 gene with the original chicken and duck RPS14 genes is more than 90%, the homology difference with goats, mice and rabbits is large (between 80% and 83%), and the expression level of the RPS14 in various tissues of the broiler chicken and the broiler ducks is obviously higher than that of goats, mice and rabbits (p is less than or equal to 0.01), so that the prepared rabbit anti-chicken RPS14 protein polyclonal antibody has good specificity and sensitivity and can be used for researching the positioning of broiler chicken lung, heart, pulmonary artery, liver and kidney RPS14 protein expression.

2.4 statistical treatment

Experimental data were processed using SPSS 18.0 software, the data were measured as mean ± standard deviation (M ± SD), analysis of variance was used for comparisons between groups, and P <0.05 indicated significance of difference.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A preparation method of a chicken RPS14 gene polyclonal antibody is characterized by comprising the following steps: (1) taking chicken cDNA as a template, carrying out PCR amplification of RPS14 gene to obtain a target fragment, wherein the nucleotide sequence of the primer amplified by the PCR is shown as SEQ ID NO: 1-2; (2) connecting the target fragment with a PET-28a (+) expression vector to construct a recombinant expression plasmid PET28a-RPS 14; (3) transferring the recombinant expression plasmid PET28a-RPS14 into a Rosetta (DE3) competent cell, performing prokaryotic expression, purifying, and performing dialysis renaturation to obtain chicken RPS14 recombinant protein; (4) taking blood after immunizing rabbits with the chicken RPS14 recombinant protein, and separating serum to obtain the chicken RPS14 gene polyclonal antibody; (5) and performing antibody quality verification on the chicken RPS14 gene polyclonal antibody by using enzyme-linked immunosorbent assay, protein immunoblotting detection or immunofluorescence technology.

2. The preparation method according to claim 1, wherein the PCR amplification in step (1) is performed by: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 35s, and extension at 72 ℃ for 40s for 35 cycles; extension at 72 ℃ for 10 min.

3. The preparation method according to claim 1, wherein the step (3) of prokaryotic expression comprises the following specific steps: to Rosetta (DE3) competent cells, which were transferred to PET28a-RPS14, IPTG was added to a final concentration of 1mM and shaken for 4h at 37 ℃ on a shaker at 220 rpm/min.

4. The method according to claim 1, wherein the eluate purified in the step (3) is an eluate having an imidazole concentration of 500 mM.

5. The method according to claim 1, wherein the dialysis renaturation of step (3) is carried out by dialyzing the target protein with urea gradient descent, and the dialyzate is replaced every 4h in an environment of 4 ℃.

6. The preparation method of claim 1, wherein the specific method for immunizing rabbits with the chicken RPS14 recombinant protein in step (4) is as follows: the chicken RPS14 recombinant protein was injected subcutaneously in New Zealand white rabbits at multiple sites to elicit an immune response, which was boosted every 10 days for a total of 30 days.

7. The method of claim 1, wherein the optimal dilution concentration of the antigen coating in the ELISA assay in step (5) is 2.5 μ g/ml; the dilution gradient of both the anti-RPS 14 serum and the pre-immune serum was 1:100 to 1: 204800; the dilution concentration of goat anti-rabbit IgG labeled by horseradish peroxidase is 1: 3000A; the reaction wavelength of the microplate reader is 450 nm;

8. a chicken RPS14 polyclonal antibody prepared according to the preparation method of any one of claims 1-7.

9. The use of the chicken RPS14 polyclonal antibody according to claim 8 in the preparation of a tool for detecting chicken pulmonary hypertension and/or ascites diseases.

10. Use according to claim 9, wherein the means is a reagent or kit.