CN112812181A

CN112812181A - Chicken JAZF1 gene polyclonal antibody and preparation method and application thereof

Info

Publication number: CN112812181A
Application number: CN202110191436.3A
Authority: CN
Inventors: 刘平; 胡国良; 郭小权; 顾玥明; 刘佩; 许兰娇; 程素芳; 李麟; 李谷月; 庄煜; 杨帆; 胡睿铭; 黄橙; 黄露佳; 郭凤萍; 李庆庆; 方玮乐
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2021-05-18

Abstract

The invention discloses a chicken JAZF1 gene polyclonal antibody, a preparation method and application thereof, and belongs to the technical field of biology. The method comprises the following steps: step 1: biologically analyzing and screening a high-quality nucleotide sequence for a chicken-derived JAZF1 gene to construct a recombinant plasmid; step 2: constructing JAZF1 recombinant plasmid, preparing JAZF1 recombinant protein, and taking the recombinant protein as an antigen; and step 3: the chicken JAZF1 gene polyclonal antibody is prepared by antigen immune reaction. The method can prepare high-quality polyclonal antibody, and the polyclonal antibody can be used for detecting JAZF1 protein in animal organism, has the advantages of wide application range, strong specificity, simple operation, low cost and the like, and lays a material foundation for research and diagnosis of pathological mechanisms of various broiler chickens and other poultry diseases.

Description

Chicken JAZF1 gene polyclonal antibody and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, relates to a chicken JAZF1 gene polyclonal antibody, a preparation method and application thereof, and particularly relates to a chicken JAZF1 gene polyclonal antibody related to occurrence and development of diseases such as broiler pulmonary hypertension (ascites) and the like, and a preparation method and application thereof.

Background

Broiler Ascites Syndrome (AS) is a serious noninfectious metabolic mass-triggered poultry disease with major clinical pathological symptoms of listlessness, white crowned flesh, dyspnea, abdominal swelling in deep purple color, white green loose feces discharge and the like, and is also called broiler pulmonary hypertension syndrome and heart failure syndrome. The diseases are mainly caused by factors such as nutrition, environment, heredity, feeding management and the like, and are highly developed in a fast-growing young chicken flock because the chicks grow rapidly, nutrition metabolism is vigorous, and the balance between biological oxygen demand and oxygen supply is destroyed, so that a large amount of oxygen-carrying red blood cells cannot smoothly flow out in capillaries of the lung, blood perfusion to the lung is inhibited, pulmonary arterial pressure is continuously increased, and finally the heart failure is caused to form ascites. Research has shown that the pulmonary hypertension and ascites diseases cause serious harm to the commercial broiler breeding industry in China, the broiler diseases generally occur in cold winter and spring with obvious mass-sending property, the morbidity can reach 10% -40%, the mortality can reach 10% -20%, and the mortality can reach 60% in serious conditions, and the broiler diseases are in a trend of rising year by year. At present, the diseases are reported in succession in other countries such as the United states, Canada, British and the like, the morbidity of the diseases is about 4 percent worldwide, the mortality can reach about 2-5 percent, the annual economic loss reaches hundreds of millions of dollars, and obviously the diseases seriously damage the broiler breeding industry all over the world.

Zinc Finger parallel gene 1(Juxtaposed with other Finger gene 1, JAZF1) also known as Tip27(TAK1-interacting protein 27) is involved in regulating various biological activities of the body. The existing research shows that the expression of JAZF1 can reduce the accumulation of lipid by enhancing the degradation of lipid, regulate the expression of a key enzyme phosphoenolpyruvate carboxylase (PEPCK) to regulate gluconeogenesis, further control the carbohydrate and lipid metabolism of an organism, and prevent the obvious hypoxia caused by insufficient perfusion of the heart and lung blood of the organism and further the injury of arterial vascular endothelial cells due to the obstruction of the blood flow caused by the massive fat deposition of the pulmonary artery. Meanwhile, as an auxiliary inhibiting factor of TAK1, the gene can inhibit NF-kB signal channels, weaken proliferation and diffusion of abnormally damaged cells, promote a large amount of apoptosis of the abnormally damaged cells and further play a multi-effect protection role on animal organisms. In conclusion, JAZF1 has a close relationship with diseases such as pulmonary hypertension and ascites of broiler chickens.

In the preliminary research in the laboratory, Yang et al have proved that the expression of the mRNA of the JAZF1 gene is reduced when broilers suffer from the diseases compared with the normal group. This indicates that at the molecular level, expression of JAZF1 has a significant impact on the development of this disease. However, the effect on such diseases at the protein level has not been clear. At present, the research on regulating the growth of organisms and improving symptoms such as diabetes and the like by regulating the expression of JAZF1 is more, but the detection and pathological mechanism research of ascites diseases of broiler chicken by preparing JAZF1 polyclonal antibody is hardly reported.

Disclosure of Invention

The invention aims to provide a chicken JAZF1 gene polyclonal antibody, a preparation method and application thereof, which are used for solving the problems in the prior art, the polyclonal antibody is simple and convenient to prepare, low in cost, high in sensitivity and wide in action range, and provides material support for later detection of JAZF1 protein and research on ascites disease pathological mechanisms.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a preparation method of chicken JAZF1 gene polyclonal antibody, which comprises the following steps:

step 1: biologically analyzing and screening a high-quality nucleotide sequence for a chicken-derived JAZF1 gene to construct a recombinant plasmid;

step 2: constructing JAZF1 recombinant plasmid, preparing JAZF1 recombinant protein, and taking the recombinant protein as an antigen;

and step 3: the chicken JAZF1 gene polyclonal antibody is prepared by antigen immune reaction.

Preferably, the nucleotide sequence of the JAZF1 gene is as shown in SEQ ID NO: 1, and the coded amino acid sequence is shown as SEQ ID NO: 2, respectively.

Preferably, the JAZF1 recombinant protein comprises the amino acid sequence as set forth in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Preferably, in the step 2, the specific method is as follows:

s1: obtaining a JAZF1 gene sequence, and constructing a PET32a-JAZF1 recombinant expression vector;

s2: and transferring the PET32a-JAZF1 recombinant expression vector into escherichia coli to induce and express JAZF1 recombinant protein, and obtaining JAZF1 recombinant protein after sorting and purification.

Preferably, the preparation method of the chicken JAZF1 gene polyclonal antibody comprises the following steps: immunizing a New Zealand white rabbit with the antigen, and separating and purifying serum from the immunized New Zealand white rabbit to obtain the polyclonal antibody.

The invention also provides a chicken JAZF1 gene polyclonal antibody prepared by the preparation method of the chicken JAZF1 gene polyclonal antibody.

The invention also provides application of the chicken JAZF1 gene polyclonal antibody in preparation of medicaments for treating broiler pulmonary hypertension and ascites diseases.

The invention also provides application of the chicken JAZF1 gene polyclonal antibody in preparation of a detection reagent for detecting broiler pulmonary hypertension and ascites diseases. Further, the invention is not limited to the diseases of pulmonary hypertension and ascites of broiler chicken, and also comprises other diseases related to JAZF1 gene of poultry.

The invention discloses the following technical effects:

the invention adopts cloning and subcloning technologies to construct a PET32a-JAZF1 expression vector, and the recombinant JAZF1 protein expressed by isopropyl-beta-D-thiogalactoside (IPTG) induction is purified and then used as an antigen to immunize a New Zealand white rabbit, thereby preparing a high-quality polyclonal antibody. The detection method of enzyme-linked immunosorbent assay, protein immunoblotting, indirect immunofluorescence technology and the like is utilized to detect the expression of JAZF1 gene so as to verify the sensitivity and specificity of the polyclonal antibody, the result shows that the anti-JAZF 1 serum titer of the immunized New Zealand white rabbit is as high as 1:102400, the polyclonal antibody can be specifically combined with JAZF1 protein in animal tissues, and the expression of JAZF1 protein is mainly concentrated in cytoplasm parts of various organs of animals, so that the antibody is fully proved to have the advantages of good sensitivity, strong specificity, wide application range and the like, and the substance support is provided for the detection of ZF JA 1 protein and the research of ascites disease pathological mechanism.

Drawings

FIG. 1 is a technical scheme diagram of a JAZF1 gene polyclonal antibody development method related to occurrence and development of broiler pulmonary hypertension and ascites diseases;

FIG. 2 shows the results of construction of cloning and expression recombinant plasmids; a: polymerase chain reaction amplification product of JAZF1 gene (669bp), lane 1, 2: PCR amplification products of JAZF1 gene protein; lane 3: negative control; b: PCR result of the cloned recombinant plasmid, lanes 1-3:

-the JAZF1 gene fragment already inserted in the T3-JAZF1 recombinant plasmid; lane 4: negative control; c: the PCR result of the bacterial liquid of the expression vector PET32a-JAZF1 is shown in lanes 1 and 2: PET32a-JAZF1 expresses a target fragment inserted into a recombinant plasmid; lane 3: negative control; d: BamHI and HindIII double restriction enzyme identification of recombinant plasmids PET32a-JAZF1 and PET-32a empty plasmid, lane 1: recombinant plasmid PET32a-JAZF1, white arrow indicates the target band after double enzyme digestion; lane 2: PET-32a no-load plasmid; m is DNA marker (2000,5000);

FIG. 3 determination of nucleotide and amino acid sequences of the recombinant plasmid PET32a-JAZF1 (including His tag, BamHI and HindIII endonuclease portions), black underlines of the DNA and amino acid sequences of the JAZF1 gene fragment inserted into the PET-32a vector, and the non-underlined portion of the vector PET-32a (+);

FIG. 4 shows the induced expression and purification of recombinant JAZF1 protein; a: IPTG-induced expression of recombinant JAZF1 protein, lanes 1, 2: induced total protein of PET32a-JAZF1, white arrow indicates target protein; lane 3: non-induced total protein of PET32a-JAZF 1; b: identification of the expression form of the recombinant JAZF1 protein, lane 1: supernatant of PET32a-JAZF1 protein, target protein indicated by white arrow; lane 2: precipitation of PET32a-JAZF1 protein; lane 3: non-induced total protein of PET32a-JAZF 1; detection of optimal imidazole concentration of recombinant protein purification elution buffer, lanes 1-4: elution buffers of different imidazole concentrations (500mM, 400mM, 300mM, 200mM) elute the protein of interest, white arrows indicate the protein of interest bands; d: purifying the target protein by using a nickel column; lanes 1-3: purified recombinant JAZF1 total protein (30 kDa); lane 4: elution buffer, white arrow indicates the protein of interest band; lane M: a standard protein Marker (10kDa-180 kDa);

FIG. 5 is a graph of the titer of antisera prepared by indirect enzyme-linked immunosorbent assay; x-axis represents dilution gradient of anti-JAZF 1 serum; the Y-axis represents the absorbance value of the polyclonal antibody at a wavelength of 450 nm; antiserum 1-antiserum 3 respectively represent anti-JAZF 1 sera generated by 3 different New Zealand white rabbits, and the control is the light absorption value of the sera before immunization under the same dilution gradient;

FIG. 6 is a method for detecting white feather broiler JAZF1 protein by an immunoblotting method; a: expression levels of broiler chicken JAZF1 protein in five related tissues; b: JAZF1 histogram compared to β -actin expression level, X-axis represents five different tissues of pulmonary artery, liver, kidney, heart and lung, Y-axis represents JAZF1 compared to β -actin expression level; c: protein imprinting method for detecting the specificity of JAZF1 protein in prokaryotic expression, wherein the upper layer lanes 1-3: total protein (30kDa) of broiler pulmonary artery JAZF1, target protein indicated by black arrow, lower lane 1-3: negative control of pre-immune serum;

FIG. 7 shows immunofluorescence assay for expression of JAZF1 protein in meat chicken, meat duck, goat, mouse and rabbit-related tissues (pulmonary artery, liver, kidney, heart and lung); a to F: respectively representing immunofluorescence result graphs of relevant tissue slices in normal broiler chickens, ascites broiler chickens, normal meat ducks, normal goats, normal mice and normal rabbits after incubation with PBS buffer (blank control), preimmune serum (negative control) and anti-JAZF 1 serum (normal group or ascites group); blue fluorescence: nuclear staining, red fluorescence: cytoplasmic staining; g: average fluorescence intensity histogram of chicken pulmonary artery, liver, kidney, heart and lung tissue JAZF1 protein, X axis: relevant tissue, Y-axis: mean fluorescence intensity of JAZF1 protein; h: a result graph of homology comparison of JAZF1 genes of different species; i: JAZF1 protein mean fluorescence intensity histogram for different animal-related tissues, X-axis: pulmonary artery, liver, kidney, heart and lung, Y-axis: mean fluorescence intensity of JAZF1 protein.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Reagents or materials used in the following examples are commercially available, unless otherwise specified.

Based on genetic engineering and immunological knowledge, the inventors designed a technical scheme implementation route of the invention, which is specifically shown in fig. 1. The implementation of this solution will be described below in the form of an example.

Example 1 method for preparing broiler chicken JAZF1 gene polyclonal antibody

1. Construction of PET32a-JAZF1 recombinant expression vector

1.1 according to the nucleotide sequence of the chicken JAZF1 gene reported in Genbank, screening a high-quality nucleotide coding sequence through bioinformatics analysis, designing and synthesizing a specific primer as shown in a table 1, and correspondingly combining with a template to amplify the JAZF1 gene, wherein the total PCR reaction system of the JAZF1 gene is 20 mu l, and the table 2 shows.

TABLE 1 PCR reaction primers

TABLE 2 PCR reaction Overall System

PCR reaction procedure: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 61.8 ℃ for 35s, extension at 72 ℃ for 40s, 35 cycles, and finally extension at 72 ℃ for 10 min.

As is clear from FIG. 2A, based on the chicken JAZF1 gene sequence published on GenBank, a pair of primers with strong specificity is designed to amplify the coding sequence of JAZF1 gene, and a clear single specific band can be seen at the position with the molecular weight of about 669 bp.

1.2 inserting the amplified target fragment into a plasmid by TA cloning

-T3 vector, thereby obtaining recombination

-T3-JAZF1 cloning vector.

As is apparent from FIG. 2B, successful insertion of the purified target sequence into the plasmid was confirmed by the PCR result of the plasmid

-T3 vector and transferred into DH5 alpha competent cells to amplify the recombinant plasmid.

1.3 carrying out double enzyme digestion on the recombinant cloning vector and the PET-32a (+) expression vector, and connecting the obtained target fragment with the PET-32a (+) expression vector to construct the PET32a-JAZF1 recombinant expression vector.

As is apparent from FIGS. 2C-D, the double digestion identification analysis of the bacterial liquid PCR and the recombinant plasmid proves that under the original amplification condition, the target fragment in the recombinant plasmid can be amplified by using the known primer, and after the recombinant plasmid is double digested, an obvious target band appears at the position with the corresponding size, and the recombinant expression vector map of FIG. 3 is combined, thereby showing that the PET32a-JAZF1 expression vector is successfully constructed.

2. Acquisition of anti-JAZF 1 protein

2.1 the successfully constructed PET32a-JAZF1 expression vector is transferred into escherichia coli DH5 alpha competent cells, and is shaken for 10h at 20 ℃ and on a shaking table at 180rpm/min by using IPTG with the final concentration of 1mM, and escherichia coli expression recombinant protein is induced and cultured. The obtained recombinant protein was subjected to 12% SDS-PAGE to identify whether it was the desired target protein.

As can be seen from fig. 4A, recombinant JAZF1 protein was expressed in large amounts under this condition and indicated by white arrows. And a large amount of JAZF1 protein appeared mainly in the supernatant of E.coli culture broth, as shown in FIG. 4B.

2.2 the expressed JAZF1 recombinant protein is purified by a nickel affinity column, elution conditions are optimized by elution buffers with the concentrations of 500mM, 400mM, 300mM and 200mM of imidazole respectively, the optimal elution concentration is determined to be 500mM of imidazole by 12% SDS-PAGE electrophoresis, and the purified recombinant protein is stored for later rabbit immunization.

As shown in FIGS. 4C-D, the elution effect of different imidazole elution concentrations is less, but the 500mM elution band is the most clear and single, the elution buffer solution with 500mM imidazole concentration is used for eluting the protein, and the JAZF1 recombinant protein is purified by a nickel affinity column to obtain the protein with the molecular weight of 30kDa, which is consistent with the expected size.

2.3 mixing the purified JAZF1 recombinant protein with Freund's complete adjuvant in equal volume, carrying out subcutaneous multipoint injection on New Zealand white rabbits, carrying out boosting immunization once every 10 days (boosting immunization requires mixing the JAZF1 protein with Freund's incomplete adjuvant), taking the ear artery blood after 30 days of immunization, and centrifuging to obtain immune serum.

3. Quality verification method of JAZF1 gene polyclonal antibody

3.1 enzyme-linked immunosorbent assay

The purified JAZF1 recombinant 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 h. Washing with PBS-T for 3 times, adding 200 μ L of 5% skimmed milk powder blocking solution to each well, incubating at 37 deg.C for 2h, washing, adding serially diluted (1:100-1:204800) anti-JAZF 1 serum and preimmune serum (100 μ L/well) with the same dilution gradient, incubating at 37 deg.C for 1h, washing, adding the mixture according to the following formula 1: 3000 diluted horseradish peroxidase-labeled goat anti-rabbit IgG (120 mu L/hole) is incubated for 1h, finally, PBS-T is used for thorough cleaning, soluble single-component TMB substrate solution is added for light-shielding reaction for 20min, concentrated sulfuric acid is used for stopping the reaction, and the absorbance value of 450nm is taken in an enzyme-labeling instrument for measurement, so that the actual titer of the JAZF1 polyclonal antibody is calculated.

As is clear from FIG. 5, the previous experiments showed that the optimal antibody titer was obtained by using an antigen coating concentration of 2.5. mu.g/ml, according to OD_{Positive for}/OD_{Negative of}The calculation formula of more than or equal to 2.1 shows that the serum titer of the anti-JAZF 1 extracted from 3 different New Zealand white rabbits is as high as 1: 102400.

3.2 Western Blot assay

After extracting total proteins of five related tissues (pulmonary artery, lung, heart, liver and kidney) of broilers in a normal group and a pathological group, measuring the protein concentration by a BCA method, performing 12% SDS polyacrylamide gel electrophoresis on 40 mu g of protein, taking a stably expressed gene beta-actin as an internal reference, transferring an electrophoresis strip onto a polyvinylidene fluoride (PVDF) membrane by wet transfer, sealing the PVDF membrane in 5% skimmed milk powder at room temperature for 2h, then incubating on a shaking table at 4 ℃ overnight with anti-ZF 1 serum (1: 3000 diluted), washing 3 times with TBS-T (10 min for each washing), incubating the membrane with goat anti-rabbit IgG (1: 1000 diluted) marked by horseradish peroxidase at room temperature for 1h, finally washing for 3 times, performing protein strip coloration by a high-sensitivity ECL chemiluminescence detection kit, and further detecting the protein level of the gene in the tissues.

As is apparent from FIGS. 6A-B, anti-JAZF 1 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 analyzing that the expression level of JAZF1 protein in the ascites group in the five tissues shows a descending trend, and the expression quantity of JAZF1 protein in pulmonary artery, heart and lung of the ascites chicken is lower than that of a control group (p is less than or equal to 0.01) by a very significant difference.

Meanwhile, anti-JAZF 1 positive serum and pre-immune serum are used as primary antibodies to carry out specificity detection on the JAZF1 protein expressed by pronucleus. As shown in FIG. 6C, the result shows that the serum before immunization can not be combined with the JAZF1 protein expressed by pronucleus, while the PVDF membrane incubated by the anti-JAZF 1 positive serum has obvious protein expression, which indicates that the polyclonal antibody has obvious reactogenicity to the JAZF1 protein.

3.3 Indirect immunofluorescence detection

Five related tissues (pulmonary artery, lung, heart, liver and kidney) of broiler chicken, meat duck, rabbit, goat and mouse are sampled and stored in 4% paraformaldehyde solution, so that the preparation of corresponding slices at the later stage is facilitated. After antigen retrieval, dewaxed slides about 5 μm thick were blocked in goat serum for 30 min. The slides were incubated overnight at 4 ℃ with Anti-JAZF 1 protein antibody (1:200 dilution) and preimmune serum (1:200 dilution), washed 3 times with PBS-T (5 min each), and incubated with Cy3 conjugated coat Anti-Rabbit IgG (H + L) incubation solution (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), adding 4',6-diamidino-2-phenylindole (DAPI; 1:400 dilution), 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. 7A-F, the PBS buffer, preimmune serum, and anti-JAZF 1 serum were used as primary antibodies to incubate 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 fluorescent signals and had strong positive compared to the two negative controls, indicating that the polyclonal antibody can specifically bind to JAZF1 protein in animal tissues, and expression of JAZF1 protein was mainly concentrated on the cytoplasmic portions of various organs of animals.

As shown in FIG. 7G, the statistical analysis of the fluorescence intensity of the broiler normal group and the ascites group revealed that the expression level of JAZF1 protein in the broiler related tissues in the normal group was significantly different from that in the ascites group (p.ltoreq.0.01).

As shown in FIG. 7H, I, the homology comparison of the chicken JAZF1 gene and the chicken JAZF1 gene is more than 90%, the homology difference with goat, mouse and rabbit is large (between 70% and 83%), and the expression quantity of the JAZF1 gene in various tissues of the chicken is obviously higher than that of goat, mouse and rabbit (p is less than or equal to 0.01), so that the prepared anti-JAZF 1 protein polyclonal antibody has good specificity and sensitivity and can be used for researching the positioning of chicken lung, heart, pulmonary artery, liver and kidney JAZF1 protein expression.

The experimental data are processed by SPSS 25.0 software, the measurement mode is expressed by mean plus or minus standard deviation (M plus or minus SD), the variance analysis is used for comparing groups, p is less than 0.05 to express the significance of the difference, and p is less than or equal to 0.01 to express the significance of the 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.

Sequence listing

<110> university of agriculture in Jiangxi

<120> chicken JAZF1 gene polyclonal antibody, preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcctccttct tctccaacgc ctgcaggttc gggggctgcg ggctgcactt cccgaccctg 60

gcggagctca tcgagcacat cgaggacaac cacatcgata cagacccacg tgtattagag 120

aaacaagaac ttcagcagcc aacctatgtt gctctaagtt acattaacag gtttatgact 180

gatgcagctc gacgtgagca ggagtctttg aagaagaaga ttcagccgaa actctctttg 240

actttgtcaa gcacggtgtc ccgtgggaat gtatccactc cacctcgcca cagcagtgga 300

agccttactc ctcccgtcac ccctccaatc actccttcct cttcatttcg tagcagtact 360

ccaacaggta gtgaatatga tgaggaggaa gtagactatg aagaatctga cagcgatgag 420

tcctggacta cagaaagtgc tatcagctct gaagctatcc ttagttcaat gtgcatgaat 480

ggcggggacg agaaaccttt cgcctgtcct gttcctggat gtaaaaaaag atataagaat 540

gtgaacggca tcaagtacca cgccaagaat ggccacagaa cacaaatccg cgtacgcaaa 600

cccttcaaat gtcgctgcgg aaagagttac aagacagctc agggcctgcg gcaccacaca 660

atcaatttc 669

<210> 2

<211> 223

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ala Ser Phe Phe Ser Asn Ala Cys Arg Phe Gly Gly Cys Gly Leu His

1 5 10 15

Phe Pro Thr Leu Ala Glu Leu Ile Glu His Ile Glu Asp Asn His Ile

20 25 30

Asp Thr Asp Pro Arg Val Leu Glu Lys Gln Glu Leu Gln Gln Pro Thr

35 40 45

Tyr Val Ala Leu Ser Tyr Ile Asn Arg Phe Met Thr Asp Ala Ala Arg

50 55 60

Arg Glu Gln Glu Ser Leu Lys Lys Lys Ile Gln Pro Lys Leu Ser Leu

65 70 75 80

Thr Leu Ser Ser Thr Val Ser Arg Gly Asn Val Ser Thr Pro Pro Arg

85 90 95

His Ser Ser Gly Ser Leu Thr Pro Pro Val Thr Pro Pro Ile Thr Pro

100 105 110

Ser Ser Ser Phe Arg Ser Ser Thr Pro Thr Gly Ser Glu Tyr Asp Glu

115 120 125

Glu Glu Val Asp Tyr Glu Glu Ser Asp Ser Asp Glu Ser Trp Thr Thr

130 135 140

Glu Ser Ala Ile Ser Ser Glu Ala Ile Leu Ser Ser Met Cys Met Asn

145 150 155 160

Gly Gly Asp Glu Lys Pro Phe Ala Cys Pro Val Pro Gly Cys Lys Lys

165 170 175

Arg Tyr Lys Asn Val Asn Gly Ile Lys Tyr His Ala Lys Asn Gly His

180 185 190

Arg Thr Gln Ile Arg Val Arg Lys Pro Phe Lys Cys Arg Cys Gly Lys

195 200 205

Ser Tyr Lys Thr Ala Gln Gly Leu Arg His His Thr Ile Asn Phe

210 215 220

Claims

1. A preparation method of chicken JAZF1 gene polyclonal antibody is characterized by comprising the following steps:

2. The method for preparing chicken JAZF1 gene polyclonal antibody as claimed in claim 1, wherein the nucleotide sequence of the JAZF1 gene is shown as SEQ ID NO: 1, and the coded amino acid sequence is shown as SEQ ID NO: 2, respectively.

3. The method for preparing chicken JAZF1 gene polyclonal antibody as claimed in claim 1, wherein JAZF1 recombinant protein contains the amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

4. The method for preparing chicken JAZF1 gene polyclonal antibody as claimed in claim 1, wherein in the step 2, the specific method is as follows:

5. The method for producing chicken JAZF1 gene polyclonal antibody as claimed in claim 1, wherein the method for producing chicken JAZF1 gene polyclonal antibody comprises: immunizing a New Zealand white rabbit with the antigen, and separating and purifying serum from the immunized New Zealand white rabbit to obtain the polyclonal antibody.

6. A chicken JAZF1 gene polyclonal antibody prepared by the preparation method of the chicken JAZF1 gene polyclonal antibody as described in any one of claims 1-5.

7. The application of the chicken JAZF1 gene polyclonal antibody in preparing medicines for treating pulmonary hypertension and ascites of broiler chickens, which is disclosed in claim 6.

8. The application of the chicken JAZF1 gene polyclonal antibody of claim 6 in preparing a detection reagent for detecting broiler chicken pulmonary hypertension and ascites diseases.