A soybean MS2 antigen, its monoclonal antibody and applications

Abstract

The present invention discloses a soybean MS2 antigen, its monoclonal antibody and applications. The present invention provides a soybean MS2 antigen. Using the soybean MS2 antigen of the present invention, a soybean MS2 monoclonal antibody that can specifically recognize exogenous and endogenous soybean MS2 proteins can be prepared. The prepared monoclonal antibody has strong specificity and a simple preparation method. In addition, using the soybean MS2 monoclonal antibody of the present invention, the MS2 protein in wild-type soybean materials can be well recognized. The monoclonal antibody can be used to assist in screening or identifying soybean MS2 protein mutants, and can also be used to analyze the interacting proteins of MS2 and its directly regulated downstream genes, for preparing stable soybean genic male sterile lines and constructing a third-generation soybean hybrid breeding system.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, it relates to a soybean MS2 antigen, its monoclonal antibody and applications. Background Art

[0002] Heterosis refers to the phenomenon that the first generation of hybrids is superior to their parents in terms of biomass, growth rate and fertility. Carrying out cross-breeding and utilizing heterosis is an effective way to rapidly increase crop yields. Compared with traditional breeding, cross-breeding has significantly increased the yields of crops such as rice, maize, wheat and rapeseed. The utilization of heterosis has increased the yields of these crops by 15% - 50%.

[0003] The cross-breeding method has gone through three generations of development. The first-generation cross-breeding method uses cytoplasmic male sterile lines, maintainer lines and restorer lines for the production of hybrid seeds, so this method is also called the three-line method. Since the cytoplasmic male sterile line contains a mitochondrial gene with a functional defect, a restorer line containing the Rf (restorer of male fertility) gene is needed to restore its fertility, thus producing hybrid seeds. However, due to the narrow germplasm resources of the restorer line and the low genetic diversity between the sterile line and the restorer line, the three-line method is limited in large-scale applications. The second-generation cross-breeding method is based on nuclear male sterile genes controlled by photoperiod and temperature. Only sterile lines and restorer lines are needed in the production of hybrid seeds, so this method is also called the two-line method. However, the two-line method is greatly affected by environmental factors, etc. Sterility such as photoperiod- and temperature-sensitive sterility is regulated by nuclear genes, and there is a defect of poor fertility stability. With the development of genetic engineering and plant transformation technologies, the third-generation cross-breeding method based on stable nuclear male sterile lines has emerged as the times require. The third-generation cross-breeding method uses stable nuclear sterile genes, overcomes the disadvantages of the three-line method and the two-line method, and has the advantages of a wide range of restorer line resources and stronger heterosis.

[0004] Soybean (Glycine max (L.) Merr.) is an important food crop and feed raw material. Due to the shortage of sterile line resources suitable for the production of soybean hybrids, large-scale utilization of heterosis has not been achieved yet. Exploring and cloning soybean nuclear male sterility genes and analyzing their functions can provide technical support for establishing the third-generation hybrid breeding system of soybeans. Currently, more than 20 soybean nuclear male sterility mutants have been identified, including structural sterility where both male and female gametes are fertile, partial male sterility, sterility in both male and female, male sterility and female fertility, photoperiod-sensitive male sterility, and male sterility controlled by a single dominant gene (Sun Xiaoyuan, Wang Yifan, Wang Yunhui, et al. Research progress on soybean nuclear male sterility genes [J]. Hereditas, 2021, 43(1): 14.). However, the key genes regulating fertility in most soybean nuclear male sterility mutants have not been clarified, and there is also a lack of antibodies that can be used to analyze the molecular functions of genes, which is not conducive to the construction of the third-generation hybrid breeding system of soybeans. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned existing technologies and provide a soybean MS2 antigen, its monoclonal antibody, and their applications.

[0006] The first object of the present invention is to provide a soybean MS2 antigen.

[0007] The second object of the present invention is to provide the application of the antigen in the preparation of soybean MS2 antibodies.

[0008] The third object of the present invention is to provide a soybean MS2 antibody.

[0009] The fourth object of the present invention is to provide a soybean MS2 monoclonal antibody.

[0010] The fifth object of the present invention is to provide the application of the antibody in the preparation of products that specifically recognize soybean MS2 protein.

[0011] The sixth object of the present invention is to provide the application of the antibody in assisting in the identification / screening of soybean MS2 mutants or in the preparation of products for assisting in the identification / screening of soybean MS2 mutants.

[0012] The seventh object of the present invention is to provide a kit for specifically recognizing soybean MS2 protein.

[0013] The above objects of the present invention are achieved by the following technical solutions:

[0014] Ms2 is one of the male-sterile and female-fertile mutants. The Ms2 gene is located between the Sat_190 and Scaa001 markers on chromosome 10, and the distances from the two markers are 6.9 and 9.0 cM, respectively. The present invention has previously identified that the key gene at the Ms2 male-sterile locus is Glyma.10G281800 (Glyma.10G281800 is the accession number of the said key gene in the soybean genome version Wm82.a2.v1 of the Phytozome website (https: / / phytozome-next.jgi.doe.gov)), denoted as MS2. MS2 is a key gene regulating soybean male fertility and is a member of the bHLH family; as a transcription factor, it can indirectly regulate soybean male fertility by directly regulating a series of genes related to pollen cell wall formation. Currently, there is no antibody available for specifically recognizing the soybean MS2 protein, and the soybean MS2 antibody can be used to analyze the proteins interacting with MS2 and its directly regulated downstream genes, for constructing the third-generation hybrid breeding system of soybean and providing technical support for the third-generation hybrid breeding of soybean. Therefore, the present invention designed different soybean MS2 antigens based on the soybean MS2 protein.

[0015] The present invention coupled different polypeptides derived from the MS2 protein with different carrier proteins to obtain different soybean MS2 antigens. Using the obtained antigens as immunogens, the corresponding monoclonal antibodies were respectively prepared. Through immunoblotting experiments on the obtained monoclonal antibodies, it was found that although some antigens had immunogenicity, the monoclonal antibodies obtained by immunization could not specifically recognize the soybean MS2 protein or had poor recognition effect on the endogenous soybean MS2 protein. Through continuous optimization, the present invention finally obtained a soybean MS2 antigen that can be used to prepare an antibody that can specifically recognize exogenous and endogenous soybean MS2 proteins.

[0016] The present invention provides a soybean MS2 antigen, which is obtained by coupling the polypeptide shown in SEQ ID NO.1 with a carrier protein.

[0017] Specifically, the antigen is obtained by coupling the C-terminal cysteine of the polypeptide shown in SEQ ID NO.1 with a carrier protein.

[0018] Specifically, the carrier protein is keyhole limpet hemocyanin (KLH).

[0019] Using the said soybean MS2 antigen, a soybean MS2 antibody that can be used to specifically recognize the soybean MS2 protein can be successfully prepared. Therefore, the present invention claims the application of the said soybean MS2 antigen in the preparation of soybean MS2 antibody.

[0020] The present invention also provides a soybean MS2 antibody, which is prepared from the said soybean MS2 antigen.

[0021] The present invention also provides a soybean MS2 monoclonal antibody, which is prepared from the soybean MS2 antigen.

[0022] Specifically, the monoclonal antibody is prepared by immunizing mice with the soybean MS2 antigen as an immunogen.

[0023] In view of the fact that the antibody prepared from the soybean MS2 antigen of the present invention can specifically recognize exogenous and endogenous soybean MS2 proteins. Therefore, the present invention also claims the application of the antibody in the preparation of products that specifically recognize soybean MS2 protein.

[0024] The present invention also claims the application of the antibody in assisting in the identification / screening of soybean MS2 mutants or in the preparation of products for assisting in the identification / screening of soybean MS2 mutants.

[0025] The present invention also provides a kit for specifically recognizing soybean MS2 protein, which contains the soybean MS2 antibody of the present invention.

[0026] Optionally, the antibody is a soybean MS2 monoclonal antibody prepared by immunizing mice with the soybean MS2 antigen as an immunogen.

[0027] The present invention has the following beneficial effects:

[0028] The present invention provides a soybean MS2 antigen. Using the soybean MS2 antigen of the present invention, a soybean MS2 monoclonal antibody that can specifically recognize exogenous and endogenous soybean MS2 proteins can be prepared. The prepared monoclonal antibody has strong specificity and a simple preparation method. In addition, using the soybean MS2 monoclonal antibody of the present invention, the MS2 protein in soybean wild-type materials can be well recognized. The monoclonal antibody can be used to assist in screening or identifying soybean MS2 protein mutants, and can also be used to analyze the interacting proteins of MS2 and the downstream genes directly regulated by it, for the preparation of stable soybean genic male sterile lines and the construction of a third-generation soybean hybrid breeding system. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the detection result of the serum titer after immunizing mice with the prepared soybean MS2 antigen.

[0030] Figure 2The results of the immunoblotting experiment of the prepared soybean MS2 monoclonal antibody against exogenous soybean MS2 protein; the red arrow in the figure indicates the position of the exogenous soybean MS2 protein (MS2-3HA fusion protein); 1, 2, and 3 represent 3 independent biological replicate experiments; Anti-HA and Anti-MS2 represent the corresponding antibodies, where Anti-HA is the HA tag antibody and Anti-MS2 is the prepared soybean MS2 monoclonal antibody.

[0031] Figure 3 The results of the immunoblotting experiment of the prepared soybean MS2 monoclonal antibody against endogenous soybean MS2 protein; a in the figure is a picture of fertile and sterile materials in the BC5F2 population offspring; b in the figure is a schematic diagram of the positions of the ms2 mutant, the MS2 antigen, and the bHLH domain in the MS2 protein; c in the figure is the result of the immunoblotting experiment of the prepared soybean MS2 monoclonal antibody against endogenous soybean MS2 protein, and the red arrow in the figure indicates the position of the endogenous MS2 protein; Anti-ACT is the antibody against ACT, and Anti-MS2 is the prepared soybean MS2 monoclonal antibody. Detailed implementation mode

[0032] The present invention will be further described below in conjunction with the specification drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0034] Example 1 Evaluation of soybean MS2 antigen and its immunization effect

[0035] The present invention couples different polypeptides derived from the MS2 protein with different carrier proteins to obtain different soybean MS2 antigens. Using the obtained antigens as immunogens, the corresponding monoclonal antibodies were respectively prepared. Through immunoblotting experiments on the obtained monoclonal antibodies, it was found that although some antigens have immunogenicity, the monoclonal antibodies obtained by immunization cannot specifically recognize soybean MS2 protein, or the recognition effect on endogenous soybean MS2 protein is not good.

[0036] Through continuous optimization, the present invention has obtained a soybean MS2 antigen that can be used to prepare monoclonal antibodies that can specifically recognize exogenous and endogenous soybean MS2 proteins. The antigen is obtained by coupling the C-terminal cysteine of the polypeptide (QQFNYNQHNRMKSDAAFSEEYQAGNSFLHDE, shown in SEQ ID NO.1) with the carrier protein (hemocyanin, KLH).

[0037] The present invention entrusts Beijing Huada Protein Research and Development Center Co., Ltd. to synthesize the polypeptide shown in SEQ ID NO.1, and couples the cysteine ​​C-terminus of the obtained polypeptide with hemocyanin to obtain the soybean MS2 antigen, which is recorded as MS2-KLH. At the same time, the company is commissioned to use the prepared soybean MS2 antigen (MS2-KLH) as an immunogen and the conjugate of the polypeptide shown in SEQ ID NO.1 and BSA protein (recorded as MS2-BSA) as a coating source to evaluate the immune effect. The details are as follows:

[0038] Using MS2-KLH as the immunogen, 4 SPF-grade Balb / c female mice were subcutaneously immunized for the first time at a dose of 60 μg protein / mouse; after the first immunization, three booster immunizations were performed with an interval of 14 days; the immunization dose was 60 μg / mouse for the first immunization and 30 μg / mouse for the booster immunization; one week after the third booster immunization, blood was collected from the eye sockets, coated with MS2-BSA, and the serum titer was detected by the Elisa method.

[0039] The serum titer test results of mice immunized with the prepared soybean MS2 antigen are as follows: Figure 1 As shown by Figure 1 The results show that mice immunized with the prepared soybean MS2 antigen (MS2-KLH) all produced mouse polyclonal antibodies, indicating that the soybean MS2 antigen of the present invention can be used for the preparation of soybean MS2 antibodies.

[0040] Example 2 Preparation of soybean MS2 monoclonal antibody

[0041] The preparation of the soybean MS2 monoclonal antibody of the present invention was entrusted to Beijing Huada Protein Research and Development Center Co., Ltd. and included the following steps:

[0042] 1) Antigen preparation

[0043] The synthetic peptide (QQFNYNQHNRMKSDAAFSEEYQAGNSFLHDE) was coupled to KLH via cysteine ​​C to prepare soybean MS2 antigen (MS2-KLH) for animal (mice) immunization;

[0044] 2) Animal immunization

[0045] Using the prepared antigen MS2-KLH as the immunogen, 4 SPF-grade female Balb / c mice were immunized; after the primary immunization, the mice were boosted 3 times at an interval of 14 days; the immunization dose was 60 μg / mouse for the primary immunization and 30 μg / mouse for the booster immunizations; one week after the third booster immunization, blood was collected from the orbital sinus. The mouse serum titer was detected by the Elisa method using the conjugate of the polypeptide (QQFNYNQHNRMKSDAAFSEEYQAGNSFLHDE) and BSA protein (MS2-BSA) for coating. One mouse with the highest titer was selected from the mice with the titer meeting the requirements for boosting fusion;

[0046] 3) Fusion

[0047] The titers of the 4 immunized mice could all meet the needs of subsequent experiments. One mouse with the highest titer was selected for boosting fusion; mouse spleen cells and SP2 / 0 cells were taken and fused by the PEG method; the fused cells were screened and cultured with a semi-solid medium (containing HAT; manufacturer: Sigma; product number: H0262-10VL);

[0048] 4) Screening

[0049] Coated with MS2-BSA, positive monoclonal cell lines specific only to the target protein (soybean MS2 protein) were screened out by the Elisa method, and subtype identification was carried out simultaneously;

[0050] 5) Verification

[0051] The cell supernatant was subjected to immunoblotting experiments for verification to determine the positive cell lines;

[0052] 6) Purification

[0053] The verified cell supernatant was purified to obtain the purified monoclonal antibody.

[0054] Using the soybean MS2 antigen described in the present invention to immunize mice to prepare monoclonal antibodies, a total of 7 positive clone cell lines were screened. The relevant information of the 7 obtained positive monoclonal cell lines is shown in Table 1:

[0055] Table 1

[0056] Number OD value for the immunogen Subtype 1 0.68 G2a 2 0.537 G1 5 0.604 G2a 10 0.714 G2b 12 0.406 G2a 16 0.59 G2a 17 0.74 G2b Negative 0.041 Empty 0.035 Positive 0.789

[0057] As can be seen from the results shown in Table 1, positive hybridoma cell lines of IgG can be successfully prepared using the soybean MS2 antigen (MS2-KLH) described in the present invention, indicating that the soybean MS2 antigen can be used to prepare soybean MS2 monoclonal antibodies.

[0058] Example 3 Immunodetection of Exogenous Soybean MS2 Protein by Soybean MS2 Monoclonal Antibody

[0059] To verify the recognition of the soybean MS2 monoclonal antibody prepared in Example 2 of the present invention for soybean MS2 protein, the CDS region of the MS2 gene sequence (gene accession number: Glyma.10G281800) was fused with the 3HA tag in the present invention. By constructing a recombinant expression vector, the MS2-3HA fusion protein (the nucleotide sequence encoding this fusion protein is shown in SEQ ID NO.2) was obtained through in vitro transcription and translation. Immunoblot analysis was performed on the obtained MS2-3HA fusion protein using the tag antibody of HA and the soybean MS2 monoclonal antibody prepared in Example 2 respectively. The results are as Figure 2 shown; Figure 2 The position of the exogenous soybean MS2 protein (MS2-3HA fusion protein) is indicated by the red arrow in

[0060] ; 1, 2, 3 represent 3 independent biological replicate experiments; Anti-HA and Anti-MS2 represent the corresponding antibodies, where Anti-HA is the HA tag antibody and Anti-MS2 is the prepared soybean MS2 monoclonal antibody. Figure 2 As can be seen, the MS2-3HA fusion protein band can be detected using the tag antibody of HA, and the same band can also be recognized using the prepared MS2 monoclonal antibody, indicating that the prepared soybean MS2 monoclonal antibody can specifically recognize the exogenous soybean MS2 protein, and the in vitro detection effect of the soybean MS2 monoclonal antibody is good. This result also shows that monoclonal antibodies that can specifically recognize exogenous soybean MS2 protein can be prepared using the soybean MS2 antigen described in the present invention.

[0061] Example 4 Immunodetection of the soybean MS2 monoclonal antibody for endogenous soybean MS2 protein

[0062] Since there has been no previous report on the specific candidate genes of the soybean ms2 male sterility locus. In order to map the ms2 locus, in the early stage of the present invention, an ms2 sterile material was used as the female parent, and Jiin16 (JL16) was used as the recurrent parent to obtain the BC5F2 population material. Through fine mapping analysis, the candidate gene of ms2 was determined to be Glyma.10G281800 (the accession number of this gene in the soybean genome version Wm82.a2.v1 of the Phytozome website (https: / / phytozome-next.jgi.doe.gov)), denoted as the MS2 gene. The fertile materials in the BC5F2 population offspring were named NIL-MS2 / JL16, and the sterile materials were named NIL-ms2 / JL16 (pictures of the fertile and sterile materials are as Figure 3as shown in a of Figure 3 as shown in b of

[0063] The recognition site of the soybean MS2 antigen described in the present invention is after the termination site of the ms2 mutant. Theoretically, the truncated protein sequence cannot be detected in this mutant using the prepared soybean MS2 monoclonal antibody. To verify whether the prepared soybean MS2 monoclonal antibody can specifically recognize the endogenous soybean MS2 protein, during the full-bloom stage of soybean growth, bud samples of fertile and sterile materials were respectively selected, and immunoblot analysis was performed on the MS2 protein therein.

[0064] In the present invention, early buds and pre-pollination buds of fertile material (NIL-MS2 / JL16) and sterile material (NIL-ms2 / JL16) were respectively selected. The bud samples were ground into powder with liquid nitrogen, and the total protein of the samples was extracted with protein extraction buffer. After quantifying the protein, immunoblot analysis was performed using actin (Actin, ACT) as an internal reference. The immunoblot experimental results of the prepared soybean MS2 monoclonal antibody against the endogenous soybean MS2 protein are as Figure 3 shown in c of Figure 3 As can be seen from c of Figure 3 the position where the mutant ms2 terminates is pointed out in b of

[0065] The above results are consistent with the expectations, indicating that the soybean MS2 monoclonal antibody prepared by the present invention can well recognize the MS2 protein in wild-type materials and can be used to specifically recognize the soybean MS2 protein. The above results also show that the monoclonal antibody capable of specifically recognizing the soybean MS2 protein can be prepared by using the soybean MS2 antigen described in the present invention. Using the antibody prepared by the present invention, the molecular function of MS2 can be further analyzed to construct the third-generation hybrid breeding system of soybeans.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A soybean MS2 antigen, characterized in that, The antigen is obtained by conjugating the polypeptide shown in SEQ ID NO.1 with a carrier protein.

2. The antigen according to claim 1, wherein The antigen is obtained by conjugating the C-terminal cysteine of the polypeptide shown in SEQ ID NO.1 with a carrier protein.

3. The antigen according to claim 1 or 2, characterized in that, The carrier protein is hemocyanin.

4. Use of the antigen according to any one of claims 1 to 3 in the preparation of a soybean MS2 antibody.

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