阅读说明：本技术 抗水稻育性发育蛋白OsP31多克隆抗体及其制备方法和应用 (Anti-rice fertility developmental protein OsP31 polyclonal antibody and preparation method and application thereof ) 是由 纪剑辉 周颖君 高清松 刘廷武 罗玉明 李正鹏 于 2021-01-15 设计创作，主要内容包括：本发明提供了抗水稻育性发育蛋白OsP31多克隆抗体及其制备方法和应用,属于分子生物技术领域及免疫学领域。本发明包括：(1)分析预测水稻OsP31蛋白抗原性,选择多肽蛋白序列作为免疫抗原；(2)构建含有抗原的表达载体；(3)利用所述表达载体转化大肠杆菌；(4)表达并纯化所述多肽；(4)使用所述多肽或含有所述多肽的组合物免疫哺乳动物；(5)从所述哺乳动物血清中纯化抗体,即得抗水稻育性发育蛋白OsP31抗体。本发明所提供特异性抗体,能够通过免疫荧光染色技术特异性识别水稻中育性发育蛋白OsP31,能够检测及区分水稻减数分裂、雄配子发育等过程中的相关突变体,亲和力强、特异性好,具备工业化生产的可能性。(The invention provides a polyclonal antibody against rice fertility developmental protein OsP31, a preparation method and application thereof, belonging to the field of molecular biotechnology and the field of immunology. The invention comprises the following steps: (1) analyzing and predicting the antigenicity of OsP31 protein of rice, and selecting a polypeptide protein sequence as an immune antigen; (2) constructing an expression vector containing the antigen; (3) transforming escherichia coli by using the expression vector; (4) expressing and purifying the polypeptide; (4) immunizing a mammal with said polypeptide or a composition comprising said polypeptide; (5) purifying the antibody from the serum of the mammal to obtain the anti-rice fertility development protein OsP31 antibody. The specific antibody provided by the invention can specifically identify the fertility development protein OsP31 in rice through an immunofluorescence staining technology, can detect and distinguish related mutants in the processes of rice meiosis, male gamete development and the like, has strong affinity and good specificity, and has the possibility of industrial production.)

1. An immune antigen of rice OsP31 protein, the amino acid sequence of which is shown in SEQ ID NO. 15.

2. An immunogenic composition comprising the immunizing antigen of claim 1.

3. An expression cassette, recombinant vector, recombinant microorganism or recombinant cell line comprising a nucleic acid molecule encoding the immunizing antigen of claim 1.

4. A preparation method of a polyclonal antibody against rice fertility developmental protein OsP31 is characterized in that an immune antigen of rice OsP31 protein with an amino acid sequence shown as SEQ ID NO.15 or a composition thereof is used for immunizing mammals.

5. The method for preparing the polyclonal antibody against rice fertility developmental protein OsP31 according to claim 4, wherein the steps are as follows:

(1) constructing a recombinant expression vector containing the immune antigen;

(2) preparing a recombinant microorganism or a recombinant cell line using the recombinant expression vector;

(3) expressing and purifying the immunizing antigen;

(4) immunizing a mammal with the immunizing antigen or a composition containing the immunizing antigen;

(5) and purifying the antibody from the serum of the mammal to obtain the anti-rice fertility developmental protein OsP31 polyclonal antibody.

6. The method for producing a polyclonal antibody against rice fertility developmental protein OsP31 according to claim 5, wherein the recombinant microorganism is Escherichia coli.

7. The antibody produced by the method for producing a polyclonal antibody against rice fertility developmental protein OsP31 according to any one of claims 4 to 6.

8. Use of the antibody of claim 7 for detecting the orientation or distribution pattern of the OsP31 gene in a mutant or wild-type plant cell.

9. The use of claim 8, wherein the plant is rice, corn, canola, wheat or soybean.

Technical Field

The invention belongs to the field of molecular biotechnology and immunology, and particularly relates to a polyclonal antibody against rice fertility developmental protein OsP31, and a preparation method and application thereof.

Background

Meiosis is a highly conserved cell division process in all sexual reproduction organisms. The overall process of meiosis involves one DNA replication and two consecutive divisions of the nucleus, which can be defined cytologically as meiosis I and II. During meiotic prophase I, homologous chromosome pairing, association and recombination occur, followed by segregation during late stage I, at which time the two sister chromatids are pulled to the two poles. Homologous recombination is essential for the maintenance of genetic diversity and is also a prerequisite for the normal segregation of homologous chromosomes.

The synaptonemal complex is a marker structure of meiotic prophase I, and its correct formation and dissociation are essential guarantees for homologous chromosome pairing, synaptation, recombination and isolation. Structurally, the synaptonemal complex is a zipper-like structure. The zipper axis is formed during the meiotic telogen phase and the association is followed by two parallel backbones of the zipper of the synaptonemal complex, and thus the chromosomal axis is also commonly referred to as the synaptonemal lateral element, and its proteins are commonly referred to as lateral element proteins. The assembly of the synaptonemal complex starts from the pre-meiotic stage I and from the even-linear stage, the pachytene stage tends to mature and function, and to the bi-linear stage begins to disintegrate. Studies in plants show that from the even line phase to the pachytene phase, the homologous chromosomes that make up the lateral elements are closely linked by transverse fibrin, while the central element protein is formed in the central position; by late pachytene, the synaptonemal complex will gradually disintegrate; by the time of the diploma, the synaptonemal complex has been substantially completely disintegrated while the homologous chromosomes remain cross-linked together, a process which is essential for the correct separation of the homologous chromosomes. Meiosis is important not only to keep the chromosome number of offspring constant, but also to exchange genetic material between individuals between parent chromosomes through homologous recombination, thereby maintaining genetic diversity of species and promoting evolution of species. In the case of animals and plants, meiosis, the most basic characteristic, presents an infinitely wide opportunity for genetic improvement. Rice OsP31 is a key gene (GeneID: 4338221) playing an important control role in the development process of rice pollen, and the full research of the gene can provide germplasm resources for innovation of a rice male sterile line.

With the continuous and deep research on the fertility mechanism, the expression change of related proteins in the rice meiosis process belongs to an extremely sensitive index, and researches on the distribution and change conditions of polyclonal antibodies of the genes on the cytological level are beneficial to researchers and breeders to further fully know the mechanism of rice fertility development, while the previous researches on the genes in the fertility process are limited to cytological phenotype observation, anther morphological analysis, gene expression change research and the like, and the researches only can explain the functional change of the proteins and cannot fully reflect the space-time change condition of the related specific proteins in specific tissues and can not completely reflect the change condition in the whole meiosis; furthermore, the relationship of these proteins to other meiotic fertility proteins at the cellular level is not clear.

Disclosure of Invention

The invention aims to provide a polyclonal antibody against rice fertility protein OsP 31. The invention also provides a method for preparing the polyclonal antibody against rice fertility developmental protein OsP 31. The last technical problem to be solved by the invention is to provide a specific application of the polyclonal antibody against rice fertility protein OsP 31.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

an immune antigen of rice OsP31 protein, the amino acid sequence of which is shown in SEQ ID NO. 15.

An immunological composition comprising said immunological antigen.

An expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell line comprising a nucleic acid molecule encoding said polypeptide.

A method for preparing polyclonal antibody against rice fertility developmental protein OsP31 comprises immunizing mammal with immune antigen of rice OsP31 protein with amino acid sequence shown as SEQ ID NO.15 or its composition.

Further, the method specifically comprises:

(1) constructing a recombinant expression vector containing the rice OsP31 protein immune antigen;

(2) preparing a recombinant microorganism or a recombinant cell line using the recombinant expression vector;

(3) expressing and purifying the rice OsP31 protein immune antigen;

(4) immunizing by using the immune antigen of the rice OsP31 protein or a composition containing the immune antigen of the rice OsP31 protein, wherein the immunized animal is one of rabbit, mouse, rat, goat and the like;

(5) and purifying the antibody from the serum of the mammal to obtain the anti-rice fertility developmental protein OsP31 polyclonal antibody.

Further, the recombinant microorganism is Escherichia coli.

Any one of the antibodies prepared by the preparation method of the anti-rice fertility developmental protein OsP31 polyclonal antibody.

The antibody can be applied to the application of monitoring the positioning or distribution rule of target protein in plant cells based on antigen-antibody reaction, and particularly applied to immunofluorescence staining experiments.

Further, the plant is rice, corn, rape, wheat or soybean.

Compared with the prior art, the invention has the beneficial effects that:

1) the antibody prepared by the invention has high titer, strong affinity, good specificity, low preparation cost, high yield and feasibility of industrial production, and can generate specific binding reaction with rice OsP31 protein.

2) The antibody prepared by the invention is a specific antibody which is selected from a plurality of candidate polyclonal antibodies and can be used for immunofluorescence histochemical analysis, can accurately analyze the expression conditions of rice in different development periods and under different growth conditions, can be applied to the research of all mechanism mechanisms related to rice fertility development, and provides detection and verification technical support for the research of the functions and action mechanisms of rice OsP31 protein. For example, the cellular localization of the fertility protein OsP31 in rice and other plants can be observed by methods such as immunohistochemistry, immunoblotting and the like, the mechanism for regulating and controlling the meiosis and fertility control of plants is disclosed, the internal relation between the flower development of the plants and the sexual reproduction of the plants is clarified, and thus the fertility regulation mechanism is utilized to lay theoretical and technical foundation for the genetic improvement of the plants.

3) The antibody prepared by the invention is a functional protein for specifically recognizing the antigen. The primary structure of a protein, i.e., its antigenic amino acid sequence, is primarily recognized. The antibody is used for identifying the rice OsP31 protein, the content of the protein can be accurately reflected, and the situation that the protein cannot be detected due to structural change caused by improper sample treatment or storage conditions is avoided.

Drawings

FIG. 1 is a schematic diagram showing the positions of amplification products of 7 antigens on the full-length DNA of a gene according to Table 1;

FIG. 2 is an SDS-PAGE identification electrophoresis chart of OsP31 prokaryotic expression protein purified by using antigen 7 obtained by combining primers of SEQ ID NO.13 and SEQ ID NO.14, wherein 1 is a protein marker (KDa); 2 is standard protein BSA; 3 and 4 are purified proteins; the band indicated by the arrow is the target protein band;

FIG. 3 is a Western detection diagram of a polyclonal antibody corresponding to an antigen 7, wherein 1 is a protein Marker; 2-4 for OsP31 antibody localization in wild type Nipponbare material, and 5-6 for OsP31 antibody localization in mutant material;

FIG. 4 is a graph showing the analysis of the localization signals of OsP31 polyclonal antibody immunized with antigen 7 and OsREC8 protein in wild-type pollen mother cells, and the length of the scale bar is 5 μm;

FIG. 5 is a graph showing the results of using immunofluorescence staining assay to detect the application of OsP31 antibody (polyclonal antibody obtained by immunizing antigen 7) in different rice mutants.

Detailed Description

The invention is further described with reference to specific examples. But do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

pMD19-T vector: the product catalog number of the Beijing Hua ao Zhengsheng science and technology Limited company is 3271.

PET30 carrier: pET30a (Novagen, USA, cat # 69909-3).

BL21 expression strain: (Beijing Quanjin Biotechnology Ltd., Cat. No. CD 601).

Indica 3037 in rice variety: description of the invention

"Zhongxian 3037" in "Wang, K., Tang, D., Hong, L., Xu, W., Huang, J., Li, M., Gu, M.s., Xue, Y.and Cheng, Z (2010) DEP and AFO regulated productive Habit in Rice.plos Genetics, 6" is publicly available from the institute of Genetics and developmental biology.

The rice variety nipponica: china institute of agricultural sciences, institute of crop science, center for the collection of rice germplasm resources, library number I1A 13071.

Example 1:

(1) analysis of protein structural features

According to the analysis of the hydrophilicity, the surface appearance, the flexibility and the like of the amino acid sequence, the antigenicity of the rice OsP31 protein is analyzed and predicted. According to the principle that polyclonal antibodies can recognize multiple antigenic determinants, 7 polypeptide protein sequences are selected to construct prokaryotic expression proteins as immune antigens, as shown in FIG. 1.

(2) Construction of prokaryotic expression vector

In order to obtain polyclonal antibodies corresponding to OsP31, 7 sets of primers (the primer sequences are respectively SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14) are respectively amplified by using cDNA of wild type rice Nipponbare to obtain amplification products with different sizes, the relevant primer sequences and amplification product sizes are shown in Table 1, the fragments are respectively introduced into an intermediate vector pMD19-T (simple), the fragments are correctly identified by enzyme digestion and sequencing, and then the fragments are respectively connected to a pET30a (Novagen Cat No. 69909-3) vector, and the vector is treated with restriction endonuclease-XhoI.

TABLE 1 designed 7 position polypeptide primer sequences and their amplified fragment sizes

(3) Expression and purification of prokaryotic proteins

The prokaryotic expression vector is transformed into escherichia coli BL21 competence by a heat shock method. A single colony was inoculated into 4mL of LB medium containing kanamycin and cultured overnight at 37 ℃ with shaking at 200rpm as a seed solution. Inoculating the seed solution into 400mL of fresh LB culture medium containing kanamycin according to the dilution ratio of 200 times, adding IPTG (isopropyl-propyl G concentrated thiogalactopyranoside) with the final concentration of 1mM when the OD value of thalli reaches 0.6, carrying out shaking culture at 180rpm for 12h, inducing expression, and centrifuging for 15min at 4 ℃ to collect thalli, wherein the final protein expression concentration is 1.2mM-1.6mM which is a proper concentration. PBS (containing 1% Triton-100) in a volume of 1/10 cells was added to the cells, and after mixing, the cells were subjected to ultrasonic cell disruption for 40 times at 10 seconds intervals, and the cells were kept in a low-temperature state during ultrasonication. The mixture was centrifuged at 12000rpm for 15min at 4 ℃ to remove the supernatant, and 1/20 volumes of protein Loading Buffer were added to express the formed protein as inclusion bodies, so that the protein samples were subjected to 12% SDS-PAGE gel electrophoresis, and FIG. 2 is a graph showing the results of the electrophoresis using antigen 7 as an example. And (3) cutting a specific recombinant protein strip after the KCl solution is dyed, airing and grinding into dry powder.

(4) Immunizing animals

Selecting healthy New Zealand white rabbits with the age of about three months and the body weight of more than 2kg, and immunizing animals with the antigen prepared in the step (3). The method comprises the following specific steps:

first, each rabbit was pre-immune induced, and local immune response was stimulated by subcutaneous injection of 0.5mL freund's complete adjuvant into the extremities, axilla and back, followed by a week's first immunization. Before the first immunization, blood was taken via the auricular vein as a negative control. Antigen was treated with PBS (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mM KH₂PO₄pH7.4) to 1mg/mL, subpackaged and stored at-20 ℃. 500 μ L of 1mg/mL antigen (i.e., 0.5mg) was added to 300 μ L PBS for another dilution, and then an equal volume of Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant (second to fourth immunization) was added. The rabbit was injected subcutaneously at multiple sites in the limbs, axilla and back, and the first immunization was followed by a second immunization, followed by boosting at two weeks intervals (4 immunizations in total). After 12 days of the third immunization, blood was taken from the ear vein to determine the antibody titer. The carotid artery was bled 11 days after the fourth immunization and blood samples were collected. Standing blood sample at 4 deg.C overnight or 37 deg.C, incubating for 3h, centrifuging at 4 deg.C and 5000rpm for 10min, collecting serum, packaging, and storing at-80 deg.C.

(5) Antibody Western Blotting detection

The rice material Nipponbare and the OsP31 gene mutant are selected as experimental materials, and a plant protein extraction kit (product number: CW0885B) of Kangji century company is used for respectively extracting total protein. The method comprises the following specific steps:

selecting rice leaf tissues of which the weight is about 100mg, placing the rice leaf tissues in a mortar, adding 0.5mL of extraction reagent, and homogenizing; after homogenizing, incubating on ice for 20 minutes, and centrifuging for 20 minutes by using a low-temperature high-speed refrigerated centrifuge at the temperature of 4 ℃ and the rpm of 12,000; the soluble protein in the supernatant was collected and left at 4 ℃ for further experiments. Extracting protein, performing SDS-PAGE electrophoresis, transferring to nitrocellulose membrane by semi-dry transfer apparatus, and sealing with 5% skimmed milk powder (TBST buffer) at 4 deg.C for 2 hr; adding purified polyclonal antibody (1: 5000), and reacting at 4 deg.C for 12 h; adding goat anti-rabbit IgG antibody (1: 2000) labeled with horseradish peroxide, and reacting at 37 deg.C for 1 h. And (3) displaying a detection result: as can be seen from the graph, the supernatant contained a band of the size of the protein of interest, which is a specific antigen of the rice OsP31 antibody (FIG. 3). The detection result shows that the wild rice Nipponbare has a single clear hybridization strip, and the mutant has no detection signal. The results prove that: the polyclonal antibody prepared by the invention can identify OsP31 protein in plants and can be specifically combined with OsP31 protein. Therefore, the polyclonal antibody can be used as a detection reagent of the OsP31 protein,

(6) chromosome immunofluorescence staining experiment for observing location of protein on chromosome

1) Collecting fresh young ears, and fixing with 4% paraformaldehyde (w/v) at room temperature for 30 min;

2) selecting small flowers with proper time, taking out anthers, and tabletting by using PBS buffer solution;

3) soaking in liquid nitrogen for 20s, quickly removing the cover glass with a blade, and air drying;

4) adding 50 μ L TNB buffer solution containing anti-antibody, covering with sealing film, placing into a wet box, and keeping the temperature at 37 deg.C for 1 h;

5) washing with PBS buffer solution for three times, each time for 5min, and air drying;

6) adding 50 μ L of TNB buffer solution containing TRITC-coupled goat anti-rabbit secondary antibody and FITC-coupled goat anti-mouse secondary antibody in dark condition, covering with sealing film, and placing into a wet box, and keeping the temperature at 37 deg.C for 30 min;

7) washing with 1 × PBS buffer solution for three times (5 min each time) in dark condition, and air drying;

8) adding 10 mu LDAPI staining solution under the dark condition, covering a cover glass, tabletting, and observing and photographing under a fluorescence phase contrast microscope.

FIG. 4 shows the localization signal analysis of the OsREC8 protein in wild-type pollen mother cells by polyclonal antibody corresponding to OsP31 protein. The immunofluorescence mapping results for the OsP31 antibody are shown in fig. 4, and to show morphological features of chromosomes at different periods, the Marker protein association complex lateral element protein OsREC8 was used for labeling; in the thin line phase, OsP31 appears intracellularly beginning with a punctate signal (fig. 4A), during the even line phase, OsP31 protein exhibits a discontinuous linear signal profile (fig. 4B), from the even line phase to the early pachytene phase, this linear signal is gradually increased, appears as a continuous linear signal, and its linear signal is able to co-localize with the OsREC8 protein (fig. 4C). From late pachytene to early bifilar, the signal of OsP31 protein showed gradual decline, and only part of punctate signal remained in the cells (FIG. 4D); FIG. 5 shows the immunofluorescence mapping analysis of OsP31 protein rice different types of mutants. The protein OsREC8 is used for displaying chromosome axis in meiosis stage, the protein can well identify the position of chromosome in cells (red), the green signal is used for displaying in the fluorescent staining of OsP31 antibody, and the OsP31 antibody can not be normally positioned in pollen mother cells of different types of synaptonemal complex mutants such as zep1, crc1 and OsP31 (figure 5).

From the observation of the chromosome immunofluorescence staining experiments of the obtained 7 different polyclonal antibodies (fig. 4-5), it was found that the polyclonal antibody obtained from antigen 7 can be effectively detected, can recognize the OsP31 protein in plants, and can be specifically bound to the OsP31 protein. Therefore, the polyclonal antibody obtained by the antigen 7 can be used as an OsP31 protein detection reagent and has important significance for functional identification of OsP31 protein. The amino acid sequence of the antigen 7 is shown as SEQ ID NO.15 and is a small peptide fragment, and the nucleotide sequence for coding the antigen is shown as SEQ ID NO.16 and has the length of 537 bp.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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65 70

<210> 26

<211> 210

<212> DNA

<213> Oryza sativa

<400> 26

atggagaggg ctaccacctc cggaggcggc ggcggcggca gccagccgcc gcggggggta 60

gggctgccgt tggtggaggt gcaggcggcg gcggcgtcgc tgcggcggtc ggaggtgttc 120

tacgtcgtga aggagctcct cggcttcgtc ctctacatgc accaccagat ccccgcggta 180

ttgcagaatc ttgaaaatga atttgcaagt 210

<210> 27

<211> 125

<212> PRT

<213> Oryza sativa

<400> 27

Arg Arg Arg Ile Lys Lys Gln Glu Lys Leu Met Asn Gly Leu Ser Ser

1 5 10 15

Val Phe Ser Ala Leu Gln Lys Ala Leu Asp Glu Val Pro Ser Ile Glu

20 25 30

Gly Val Leu Leu Ile Leu Gly Gly Ser Leu Val Arg Pro Leu Phe Val

35 40 45

Tyr Asp Ile Thr Ile Ser His Gly Arg Phe Asp Ala Gly Ser Ala Asn

50 55 60

Glu Arg Gly Ala Ser Lys Leu Ala Gln Ser Val Ser Arg Lys Ala Ile

65 70 75 80

Arg Ala Leu Ile Ser Ser Gly Ala Gly Ser Leu Ser Tyr Thr Gly Pro

85 90 95

Thr Lys Leu Phe Val Leu Val Arg Cys Pro Cys Thr Leu Asn Leu Pro

100 105 110

Leu Asp Phe Leu Pro Lys Arg Asp Phe Arg Tyr Ser Lys

115 120 125

<210> 28

<211> 375

<212> DNA

<213> Oryza sativa

<400> 28

agacggagga tcaagaagca ggagaagtta atgaatggcc tctccagcgt attttctgct 60

cttcagaaag cactcgatga agttcctagc attgaaggag ttctcctgat cctcggtggt 120

agccttgtca ggcctctgtt tgtctatgac attacaattt ctcatggtag atttgatgct 180

ggaagtgcca atgagcgtgg tgcaagcaaa ttagcgcagt ccgtttctcg aaaggccatt 240

cgtgctctta tatcaagtgg tgcagggagt ttatcttata caggccctac caagctgttt 300

gttctagtca gatgtccctg tacattgaac ttaccactgg acttcctgcc gaaacgtgat 360

tttcgttaca gcaaa 375