Scallop spermatogonium cell surface marker protein and application thereof in genetic breeding

Abstract

The invention provides a scallop spermatogonium surface marker protein and application thereof in genetic breeding. The amino acid sequence of the scallop spermatogonium surface marker protein is SEQ ID NO: 1. The invention also provides an application of the scallop spermatogonial cell surface labeled protein, namely an application of the scallop spermatogonial cell surface labeled protein as a specific label in labeling scallop spermatogonial cells. The invention further provides a method for detecting the scallop spermatogonium. According to the method, an immunohistochemical method is used for detecting whether the scallop spermatogonium surface marker protein exists or not. The scallop spermatogonial cell surface marker protein is obtained through screening and used as a scallop spermatogonial cell specific marker, and therefore a foundation is laid for scallop spermatogonial cell separation and in-vitro culture.

Description

Technical Field

[0001] The invention belongs to the technical field of marker proteins and genetic breeding, and specifically relates to a scallop spermatogonia surface marker protein and application thereof in genetic breeding. Background Art

[0002] Spermatogonia are essential for spermatogenesis and male reproduction. In vertebrates, there are three main types of spermatogonia, including undifferentiated, differentiating, and differentiated spermatogonia. Spermatogonial stem cells are undifferentiated spermatogonia that have the ability to self-renew and maintain spermatogenesis. Spermatogonial stem cells account for a very low proportion in adult testes (0.3% in mice, 4% in monkeys, and 22% in humans), and the primary task of their research is isolation and enrichment.

[0003] Current studies usually use Percoll gradient density centrifugation, differential adhesion or fluorescence-activated / magnetic-activated cell sorting to enrich spermatogonial stem cells. Among them, fluorescence-activated / magnetic-activated cell sorting has been shown to obtain spermatogonial stem cells with higher purity than other methods. However, the use of these two methods is limited by spermatogonial stem cell-specific surface markers. To date, surface markers of spermatogonial stem cells have been extensively studied in many vertebrate species, but a large number of spermatogonial stem cell markers are not conserved in different organisms. Therefore, surface markers of spermatogonial stem cells must be developed and screened in species of interest.

[0004] Molluscs are the second largest group of invertebrates, including more than 100,000 species. Most mollusks have a long reproductive cycle and are extremely vulnerable to natural disasters, infection by pathogenic microorganisms and other environmental threats. Isolating germline stem cells for cryopreservation and in vitro differentiation is expected to shorten the reproductive cycle of shellfish, preserve germplasm resources, and promote the sustainable development of shellfish farming. Compared with primordial germ cells of early developmental origin, spermatogonial stem cells come from adult gonads with a large number of cells and are therefore easier to isolate.

[0005] However, the current understanding of mollusk germ stem cells is still limited, germ cell manipulation techniques (such as isolation, enrichment, culture and gene editing) are still in their infancy, and there are no surface markers for the identification of spermatogonial stem cells or spermatogonia. Therefore, the development of scallop spermatogonia-specific surface markers will facilitate the characterization and manipulation of mollusk spermatogonia and provide useful screening markers for shellfish genetic breeding. Summary of the invention

[0006] The invention aims to provide a scallop spermatogonia surface marker protein and its application in genetic breeding, namely, a marker gene Fgfr discovered through screening and its application.

[0007] The present invention first provides a scallop spermatogonia surface marker protein, comprising:

[0008] 1) a protein having an amino acid sequence of SEQ ID NO: 1;

[0009] 2) A protein derived from 1) by replacing, deleting or adding one or more amino groups in 1).

[0010] A nucleotide sequence of the gene encoding the scallop spermatogonia surface marker protein is SEQ ID NO: 2.

[0011] The present invention also provides a recombinant expression vector for recombinantly expressing the scallop spermatogonia surface marker protein;

[0012] Furthermore, a nucleotide fragment with the sequence of SEQ ID NO: 4 is inserted into the recombinant expression vector.

[0013] The present invention also provides a recombinant expression engineering strain, into which the above-mentioned recombinant expression vector is transformed.

[0014] In yet another aspect, the present invention provides a use of the scallop spermatogonia surface marker protein, which is used as a specific marker in marking scallop spermatogonia.

[0015] The present invention also provides a product for marking scallop spermatogonia, which contains antibodies for detecting the surface marker protein.

[0016] In yet another aspect, the present invention provides a method for detecting scallop spermatogonia, wherein the method uses an immunohistochemical method to detect whether the above-mentioned scallop spermatogonia surface marker protein is present.

[0017] The present invention screens and obtains a scallop spermatogonia surface marker protein, which is used as a scallop spermatogonia specific marker, thereby laying a foundation for the separation and in vitro culture of scallop spermatogonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the single-cell expression pattern and cell localization map of the scallop Fgfr gene. The results show that the Fgfr gene is specifically expressed in spermatogonia.

[0019] Figure 2The graph shows the expression of recombinant protein of the extramembrane region of scallop FGFR protein under different conditions, wherein lane 1: total bacterial protein before IPTG induction; lanes 2-3: supernatant of bacterial lysate after induction with 0.2mM and 0.5mM IPTG at 4℃ for 16h; lanes 4-5: supernatant of bacterial lysate after induction with 0.2mM and 0.5mM IPTG at 8℃ for 16h; lanes 6-7: supernatant of bacterial lysate after induction with 0.2mM and 0.5mM IPTG at 12℃ for 16h.

[0020] Figure 3 This is the SDS-PAGE gel electrophoresis diagram of the purification of the recombinant protein of the extramembrane region of the scallop FGFR protein, wherein lane 1: total bacterial protein before IPTG induction; lane 2: supernatant of bacterial lysate after 0.5 mM IPTG induction at 12°C for 16 h; lane 3: flow-through of Ni-NTAResin affinity purification; lanes 4-12: wash solution; and lane 13: eluate.

[0021] Figure 4 This is the standard curve of the BCA determination of the recombinant protein concentration of the extracellular region of scallop FGFR protein, and the protein concentration is 0.55 mg / mL.

[0022] Figure 5 This is a Western Blot analysis of scallop FGFR recombinant protein antibody, wherein lane 1: Anti-his rabbit monoclonal antibody is used to detect scallop FGFR recombinant protein; lane 2: Anti-FGFR rabbit polyclonal antibody is used to detect scallop FGFR recombinant protein; lane 3: Anti-FGFR rabbit polyclonal antibody is used to detect scallop testis FGFR protein.

[0023] Figure 6 This is the immunohistochemical validation diagram of the scallop FGFR recombinant protein antibody. The results show that the scallop FGFR recombinant protein rabbit polyclonal antibody specifically detects spermatogonia.

[0024] Figure 7 This is an immunofluorescence validation image of the scallop FGFR recombinant protein antibody. The results show that the scallop FGFR recombinant protein rabbit polyclonal antibody specifically binds to the spermatogonia surface protein. DETAILED DESCRIPTION

[0025] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the equipment and experimental methods used in the present invention are all conventionally purchased reagents, equipment and methods of use.

[0026] Example 1: Screening of scallop spermatogonia surface marker protein genes

[0027] 1) Construct a single-cell transcriptome library of the gonad of scallops at the early stage of sex differentiation, and divide the cell groups after data processing. The spermatogonia group was determined by analyzing the expression patterns and functional enrichment of marker genes of different groups.

[0028] 2) Screen receptor genes from spermatogonia marker genes, obtain spermatogonia surface candidate marker Fgfr, and design and synthesize sense and antisense probes.

[0029] 3) In situ hybridization was performed in mature scallop testis samples to determine the cellular localization of the Fgfr gene. Paraffin sections of the testis were mounted on slides, rehydrated with gradient alcohol, and then incubated with 2μg / mL proteinase K in PBST (PBS containing 0.1% Tween 20) at 37°C for 15 minutes. After prehybridization in hybridization buffer at 60°C for 3 hours, hybridization was carried out overnight with 1μg / mL RNA probe at the same temperature. After removing the probe, the sections were incubated in blocking buffer (PBST and 0.5% blocking reagent) for 1 hour and incubated with 1:2000 anti-digoxigenin antibody (Roche, Germany) for 16 hours at 4°C. After being thoroughly washed with maleic acid buffer (0.1mol / L maleic acid, 0.15mol / LNaCl, 0.1% Tween-20, pH=7.5), the sections were incubated with NBT / BCIP solution (Roche, Germany) for color development. Finally, the sections were counterstained with 1% neutral red solution. Results are shown in Figure 1 .

[0030] The amino acid sequence of the extracellular region fragment protein of the Fgfr gene, a surface marker of scallop spermatogonia, obtained by screening is as follows:

[0031] NDLKVYEGDKARLRCVVKGHPRPWIAWTKNNQSLTPKQDPRYKINRNALVIDKVHRNDTAVFSCFAWNELGNISTTFTLTVITEEEEEPVEQSYAISKCNKKEEGAPIWKTSKENSRWIARPARAPVELKCQACGNPIPNITWYKNSQIIDSSVKEGKYQVDGYKL KMTDLTTEDNGNYTCMIQNRYGKLMWMYMLEVIDRNVQKPDVEGPVNTTAYIGENVKLHCKVKSDIQAHIQWVKHYQINGTYRKPDGTPYVHVIQQSTYNMTNPEILEIINVSRSDTGWYTCLVSNFAGRRFGSAWLTVEDVPGNESLSNMRHAQEAPRSSAK(SEQ ID NO:1);

[0032] The nucleotide sequence of its coding gene is as follows:

[0033] AATGATCTGAAAGTTTACGAAGGTGATAAAGCGCGTCTGCGTTGCGTTGTTAAAGGCCACCCGCGTCC

[0034] GTGGATTGCGTGGACCAAAAACAACCAGAGCCTGACCCCGAAACAAGATCCGCGTTACAAAATCAACCGTA

[0035] ACGCGCTGGTTATCGATAAAGTTCACCGTAACGATACCGCGGTTTTCAGCTGCTTCGCGTGGAACGAACTGG

[0036] GCAACATCAGCACCACCTTCACCCTGACCGTTATCACCGAAGAAGAAGAAGAACCGGTTGAACAGTCTTAC

[0037] GCGATCTCTAAATGCAACAAAAAAGAAGAAGGCGCGCCGATCTGGAAAACCAGCAAAGAAAACTCTCGTT

[0038] GGATCGCGCGTCCGGCGCGTGCGCCGGTTGAACTGAAATGCCAGGCGTGCGGTAACCCGATCCCGAACATC

[0039] ACCTGGTACAAAAACTCTCAGATCATCGATTCTAGCGTTAAAGAAGGTAAATACCAGGTTGATGGTTACAAA

[0040] CTGAAAATGACCGATCTGACCACCGAAGATAACGGTAACTACACCTGCATGATCCAGAACCGTTACGGTAAA

[0041] CTGATGTGGATGTACATGCTGGAAGTTATCGATCGTAACGTTCAGAAACCGGATGTTGAAGGCCCGGTTAAC

[0042] ACCACCGCGTACATCGGTGAAAACGTTAAACTGCACTGCAAAGTTAAATCTGATATCCAGGCGCACATCCAG

[0043] TGGGTTAAACACTACCAGATCAACGGTACCTACCGTAAACCGGATGGTACCCCGTACGTTTCACGTTATCCAGC

[0044] AGAGCACCTACAACATGACCAACCCGGAAATCCTGGAAATCATCAACGTTAGCCGTAGCGATACCGGTTGGT

[0045] ACACCTGCCTGGTTAGCAACTTCGCGGGCCGTCGTTTCGGTAGCGCGTGGCTGACCGTTGAAGATGTTCCG

[0046] GGTAACGAAAGCCTGAGCAACATGCGTCACGCGCAGGAAGCGCCGCGTAGCTCTGCGAAA (SEQ ID NO: 2).

[0047] Example 2: Preparation of scallop FGFR recombinant protein

[0048] 1) In order to prepare the extracellular region fragment protein of FGFR of SEQ ID NO: 1, a 30-aa tag peptide segment was connected to its N-terminus. The amino acid sequence of the recombinant protein to be expressed connected with the tag segment is as follows:

[0049] HHHHHHSSGLVPRGSHMASMTGGQQMGRGSNDLKVYEGDKARLRCVVKGHPRPWIAWTKNNQSLT

[0050] PKQDPRYKINRNALVIDKVHRNDTAVFSCFAWNELGNISTTFTLTVITEEEEEPVEQSYAISKCNKKEEGAPIWKTS

[0051] KENSRWIARPARAPVELKCQACGNPIPNITWYKNSQIIDSSVKEGKYQVDGYKLKMTDLTTEDNGNYTCMIQN

[0052] RYGKLMWMYMLEVIDRNVQKPDVEGPVNTTAYIGENVKLHCKVKSDIQAHIQWVKHYQINGTYRKPDGTPY

[0053] VHVIQQSTYNMTNPEILEIINVSRSDTGWYTCLVSNFAGRRFGSAWLTVEDVPGNESLSNMRHAQEAPRSSAK

[0054] (SEQ ID NO:3),

[0055] The nucleotide sequence of its corresponding coding gene is as follows:

[0056] CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGG

[0057] ACAGCAAATGGGTCGCGGATCCAATGATCTGAAAGTTTACGAAGGTGATAAAGCGCGTCTGCGTTGCGTTG

[0058] TTAAAGGCCACCCGCGTCCGTGGATTGCGTGGACCAAAAACAACCAGAGCCTGACCCCGAAACAAGATCC

[0059] GCGTTACAAAATCAACCGTAACGCGCTGGTTATCGATAAAGTTCACCGTAACGATACCGCGGTTTTCAGCTG

[0060] CTTCGCGTGGAACGAACTGGGCAACATCAGCACCACCTTCACCCTGACCGTTATCACCGAAGAAGAAGAAG

[0061] AACCGGTTGAACAGTCTTACGCGATCTCTAAATGCAACAAAAAAGAAGAAGGCGCGCCGATCTGGAAAACC

[0062] AGCAAAGAAAACTCTCGTTGGATCGCGCGTCCGGCGCGTGCGCCGGTTGAACTGAAATGCCAGGCGTGCG

[0063] GTAACCCGATCCCGAACATCACCTGGTACAAAAACTCTCAGATCATCGATTCTAGCGTTAAAGAAGGTAAATA

[0064] CCAGGTTGATGGTTACAAACTGAAAATGACCGATCTGACCACCGAAGATAACGGTAACTACACCTGCATGAT

[0065] CCAGAACCGTTACGGTAAACTGATGTGGATGTACATGCTGGAAGTTATCGATCGTAACGTTCAGAAACCGGA

[0066] TGTTGAAGGCCCGGTTAACACCACCGCGTACATCGGTGAAAACGTTAAACTGCACTGCAAAGTTAAATCTGA

[0067] TATCCAGGCGCACATCCAGTGGGTTAAACACTACCAGATCAACGGTACCTACCGTAAACCGGATGGTACCCC

[0068] GTACGTTCACGTTATCCAGCAGAGCACCTACAACATGACCAACCCGGAAATCCTGGAAATCATCAACGTTAG

[0069] CCGTAGCGATACCGGTTGGTACACCTGCCTGGTTAGCAACTTCGCGGGCCGTCGTTTCGGTAGCGCGTGGC

[0070] TGACCGTTGAAGATGTTCCGGGTAACGAAAGCCTGAGCAACATGCGTCACGCGCAGGAAGCGCCGCGTAG

[0071] CTCTGCGAAA (SEQ ID NO: 4).

[0072] The nucleic acid fragment with the sequence of SEQ ID NO: 4 synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. was connected to the pET-28a(+) vector to obtain a recombinant expression vector.

[0073] 2) The recombinant expression vector in step 1) was transformed into ArcticExpress (DE3) pRARE2 Escherichia coli (Angyu, Shanghai), and cultured overnight at 37°C in kanamycin-resistant LB solid medium. A single colony containing the pET-28a (+) -Fgfr recombinant plasmid was picked and cultured overnight in 10 mL of kanamycin-resistant LB liquid medium at 37°C and 150 rpm, then transferred to 200 mL of kanamycin-resistant LB liquid medium at a mass concentration of 1%, and cultured at 37°C and 150 rpm until the OD600 value was between 0.6 and 0.8; IPTG was added to a final concentration of 0.2 mmol / L (50 mL of bacterial solution was taken out as a control before induction), cultured at 4°C and 150 rpm for 16 h, centrifuged at 7000g for 15 min to collect the cells, discarded the supernatant, and the cells were lysed with 4 mL of non-denaturing lysis buffer (1 M NaCl, 25 mMTris-HCl, 5% glycerol, 1% Triton X-100, pH 7), resuspended, ultrasonically disrupted at 150W for 30min, supersonicated for 4s and stopped for 6s, centrifuged at 12000g for 15min, collected the supernatant and precipitate, and resuspended the precipitate in 4mL non-denaturing lysis buffer, and 40μL of sample and 10μL of 5× protein loading buffer were taken and mixed, and the mixture was incubated at 99℃ for 10min, and SDS-PAGE was performed to verify whether the protein was correctly expressed.

[0074] 3) Purifying the protein using nickel-containing affinity resin Ni-NTA Resin; after adding non-denaturing lysis buffer to equilibrate the purification column, the expression product in step 2) is washed with 0mM, 5mM, 10mM, 20mM, 50mM, 80mM, 100mM, 200mM, and 300mM imidazole in sequence to remove impurities, and then eluted with 500mM imidazole to obtain the target protein.

[0075] In order to improve the expression efficiency, in step 2), the induction conditions of IPTG final concentration of 0.2 mmol / L and induction temperature of 4°C were changed to IPTG final concentration of 0.5 mmol / L and induction temperature of 4°C; IPTG final concentration of 0.2 mmol / L and induction temperature of 8°C; IPTG final concentration of 0.5 mmol / L and induction temperature of 8°C; IPTG final concentration of 0.2 mmol / L and induction temperature of 12°C; IPTG final concentration of 0.5 mmol / L and induction temperature of 12°C.

[0076] The recombinant protein of the extracellular region of the scallop FGFR protein prepared by recombinant expression was subjected to SDS-PAGE gel electrophoresis analysis, and the voltage was adjusted to 80V for 40min and then 120V for 70min. Figure 2 and Figure 3 .

[0077] The concentration of the prepared scallop FGFR protein extracellular domain recombinant protein was determined by BCA method, and it was 0.55 mg / mL. Figure 4 .

[0078] Example 3: Western Blot Verification of Scallop FGFR Extracellular Domain Recombinant Protein Antibody

[0079] The recombinant protein obtained in Example 2 was used as an antigen for animal immunization to prepare the corresponding antibody. The rabbit polyclonal antibody was synthesized by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. The recombinant protein of the extracellular region of the scallop FGFR protein membrane in Example 2 was subjected to SDS-PAGE gel electrophoresis at a voltage of 80V for 40 minutes and then 120V for 70 minutes; a transfer system was used to adjust 300mA and transfer to a PVDF membrane for 60 minutes; non-specific sites were blocked with TBST containing 5% skimmed milk powder; the primary antibody (Anti-FGFR rabbit polyclonal antibody: TBST containing 5% skimmed milk powder 1:5000) was diluted with a blocking solution containing 5% skimmed milk powder and incubated at room temperature for 2 hours; the secondary antibody (GoatAnti-Rabbit IgG Antibody: TBST containing 5% BSA 1:5000) was diluted and incubated at 4°C overnight; an ECL chemiluminescence kit was used to perform chemiluminescence detection in an imager. The results are as follows Figure 5 shown.

[0080] Example 4: Immunohistochemical verification of scallop FGFR extracellular domain recombinant protein antibody

[0081] Paraffin sections of scallop testes were dewaxed and rehydrated, and incubated in methanol containing 3% hydrogen peroxide for 30 minutes to quench endogenous peroxidase activity. The sections were incubated in 1mmol / LEDTA at 85℃ for 40 minutes for antigen retrieval. To prevent nonspecific binding, the sections were incubated in 10% goat serum and 1% BSA in PBS. Rabbit polyclonal antibody (1:300) against the recombinant protein of the ectodomain of scallop FGFR was added to the sections and incubated overnight at 4℃. After incubation for 1 hour with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Sangon Biotech, 1:1000), the sections were stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. After dehydration with gradient alcohol, the sections were mounted and observed under a Nikon ECLIPSE Ni microscope.

[0082] See also Figure 6 The recombinant protein rabbit polyclonal antibody prepared by the present invention can specifically detect scallop spermatogonia, proving that the recombinant protein can be used as a specific marker for scallop spermatogonia.

[0083] Example 5: Immunofluorescence verification of scallop FGFR extracellular domain recombinant protein antibody

[0084] Cut the testes of growing male scallops into 1 mm 3 The cells were digested with 2 mg / mL collagenase II at 4°C for 30 minutes. The cells were evenly smeared at a concentration of 10 5 Cells / mL. The smear was fully dried at 37°C and fixed with 4% paraformaldehyde at room temperature for 10 minutes. The smear was incubated with 1% BSA in PBS at room temperature for 30 minutes, and then incubated with diluted FGFR extracellular domain recombinant protein rabbit polyclonal antibody (1:300) in 1% BSA in PBS at room temperature for 1 hour. The smear was washed with PBS 3 times, 5 minutes each time. Then ABflo 594-conjugated Goat Anti-Mouse IgG (H+L) (ABclonal, Wuhan) was diluted 1:200 and incubated at room temperature in the dark for 1 hour. The smear was washed with PBS 3 times, 5 minutes each time. The slide was sealed with anti-fluorescence quenching sealing solution containing DAPI (Biyuntian, Shanghai). Fluorescence images were obtained using a Zeiss LSM980 Airyscan2 confocal microscope.

[0085] See also Figure 7 The recombinant protein rabbit polyclonal antibody prepared by the present invention can specifically bind to the FGFR protein on the surface of scallop spermatogonia, proving that the recombinant protein can be used as a specific surface marker of scallop spermatogonia.

[0086] The above description is only a preferred specific implementation of the present invention and is not intended to limit the present invention. Any changes or substitutions within the technical scope of the present invention should be included in the protection scope of the present invention.

Claims

1. A scallop spermatogonia surface marker protein, characterized in that: The surface marker protein comprises: 1) a protein having an amino acid sequence of SEQ ID NO: 1; 2) A protein derived from 1) by replacing, deleting or adding one or more amino groups in 1).

2. A gene, characterized in that The gene encodes the scallop spermatogonia surface marker protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A recombinant expression vector for recombinantly expressing the scallop spermatogonia surface marker protein according to claim 1, characterized in that: A nucleotide fragment with the sequence of SEQ ID NO: 4 is inserted into the recombinant expression vector.

5. A recombinant expression engineering strain, characterized in that: The recombinant expression engineering strain is transformed with the recombinant expression vector described in claim 4.

6. Application of the scallop spermatogonia surface marker protein according to claim 1 as a specific marker in marking scallop spermatogonia.

7. A product for marking scallop spermatogonia, characterized in that: The product contains antibodies for detecting the scallop spermatogonia surface marker protein according to claim 1.

8. A method for detecting scallop spermatogonia, characterized in that: The method described is to use an immunohistochemical method to detect whether the scallop spermatogonia surface marker protein described in claim 1 exists to screen spermatogonia.

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