A light sea urchin growth differentiation factor-8 / 11 polyclonal antibody, antigen protein and preparation method thereof
By preparing the growth differentiation factor-8/11 antigen protein and polyclonal antibody of Echinococcus glabra, the problem of the lack of growth differentiation factor-8/11 antibody in Echinococcus glabra was solved, enabling in-depth research on its function and expression.
Patent Information
- Application Number
- CN202610408026.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-31
- Publication Date
- 2026-07-07
AI Technical Summary
The lack of antibodies against the growth differentiation factor-8/11 of *Echinochloa glabra* limits the analysis and functional study of this protein at the protein level.
The growth differentiation factor-8/11 antigen protein of sea urchin *Echinochloa glabra* was prepared, a recombinant expression vector was constructed and expressed in *Escherichia coli*, and after purification of the recombinant protein, polyclonal antibodies were obtained by immunizing mice. The expression of endogenous growth differentiation factor-8/11 was detected using this antibody.
It provides an efficient tool for the study of growth differentiation factor-8/11 in sea urchin scintillans, enabling accurate analysis of its function and expression, and filling the gap in antibodies against growth differentiation factor-8/11 in sea urchin scintillans.
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Figure CN122344245A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of antigen protein and antibody preparation technology, and in particular to a polyclonal antibody against growth differentiation factor-8 / 11 of sea urchin *Echinochloa glabra*, an antigen protein and a method for preparing the same. Background Technology
[0002] Studies in mammals have shown that growth differentiation factor-8 (GDF8, also known as myostatin) and growth differentiation factor-11 (GDF11) are two proteins in the TGF-β superfamily with highly homologous sequences but different functions. Both are synthesized as precursor proteins and, after processing, form disulfide-linked homodimers that are actively secreted proteins. GDF8 primarily functions as a muscle-specific negative regulator, while GDF11 broadly regulates various processes such as embryonic development, organ homeostasis, and aging. Furthermore, GDF11 can promote M1-to-M2 macrophage polarization and reduce inflammation.
[0003] Invertebrates do not possess independent GDF8 or GDF11 genes; they only have a single GDF8 / 11 homolog, which is the common ancestral gene of GDF8 and GDF11 in vertebrates. Currently, preliminary studies have been conducted on the function of the GDF-8 / 11 gene in invertebrates such as fruit flies, oysters, shrimp, crabs, and sea anemones. However, functional studies of the sea urchin GDF-8 / 11 gene and its protein level have not been reported. The preparation of an antibody against GDF-8 / 11 in the sea urchin *Echinochloa glabripennis* will provide a useful tool for analyzing the expression and function of GDF-8 / 11 at the protein level. Summary of the Invention
[0004] The purpose of this invention is to provide a growth differentiation factor-8 / 11 antigen protein, a polyclonal antibody, and a method for preparing the same in *Echinocactus glaucus*, filling the current gap in the lack of an antibody against growth differentiation factor-8 / 11 in *Echinocactus glaucus*.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a growth differentiation factor-8 / 11 antigen protein of sea urchin echinococcine, the amino acid sequence of which is shown in SEQ ID NO.1.
[0006] The present invention also provides the full sequence of the coding region of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene, which includes the above-mentioned coding sequence of the *Echinocactus glaucus* growth differentiation factor-8 / 11 antigen protein, and the nucleotide sequence of the coding region of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene is shown in SEQ ID NO.2.
[0007] The present invention also provides a recombinant expression vector for the growth differentiation factor-8 / 11 antigen protein of sea urchin glabra, comprising the above-mentioned coding sequence of the growth differentiation factor-8 / 11 antigen protein of sea urchin glabra and an initial vector.
[0008] Preferably, the initial vector is pET22b(+), and the coding sequence of the Echinocactus glaber growth differentiation factor-8 / 11 antigen protein is inserted between the NdeI and XhoI restriction sites of the initial vector.
[0009] This invention also provides a method for preparing the growth differentiation factor-8 / 11 antigen protein of *Echinocactus glaucus*, comprising the following steps: The above recombinant expression vector was transformed into Escherichia coli to recombinantly express the target protein, and the recombinant expressed Echinococcus glabra growth differentiation factor-8 / 11 antigen protein was obtained after purification.
[0010] The present invention also provides a polyclonal antibody against the growth differentiation factor-8 / 11 of Echinocactus glaucus, which is obtained by immunizing animals (mice) with the above-mentioned Echinocactus glaucus growth differentiation factor-8 / 11 antigen protein.
[0011] The present invention also provides a primer pair for amplifying the full sequence of the coding region of the above-mentioned Echinocactus glaucus growth differentiation factor-8 / 11 gene, the sequence of which is shown in SEQ ID NO.3-4.
[0012] Furthermore, the amino acid sequence corresponding to the full-length coding region of the antigen protein contains a signal peptide sequence and a TGF-β family domain, as well as a furin cleavage site. The sequence following the furin cleavage site is the predicted mature secretory growth differentiation factor-8 / 11 amino acid sequence, which has a conserved cysteine structure consistent with the known mature secretory growth differentiation factor-8 / 11 amino acid sequences of red sea anemones and star-shaped sea anemones.
[0013] Furthermore, the coding sequence corresponding to the secretory mature protein in the full-length coding region of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene was extracted as the insertion sequence for constructing the recombinant antigen protein expression vector. Using a plasmid obtained by TA cloning that linked the full-length coding sequence of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene as a template, the coding sequence corresponding to the secretory mature protein of *Echinocactus glaucus* growth differentiation factor-8 / 11 was inserted between the NdeI and XhoI restriction sites of the pET22b(+) restriction enzyme in the vector to construct the expression vector pET22b(+)-MnGDF8 / 11. The primer pairs used for seamless cloning are shown in Table 1. The expression vector pET22b(+)-MnGDF8 / 11 was transformed into *E. coli* Origami(DE3) using the heat shock method, and positive clones were screened. The expression of the recombinant protein Origami(DE3)-pET22b(+)-MnGDF8 / 11 was induced by IPTG, and the cells were lysed and subjected to SDS-PAGE. PAGE analysis was performed to determine the expression of the recombinant protein. The recombinant protein was obtained by nickel affinity chromatography, gradient dialysis, and concentration.
[0014] Furthermore, using the recombinant protein obtained above as an antigen, mice were immunized with an oil-in-water rapid adjuvant. The injection site was the calf muscle of the hind limb of the mouse. The mice were immunized again on day 21 after the initial immunization. Blood was collected from the eyeballs on day 35 after the initial immunization to separate serum and the antibody titer was detected by indirect enzyme-linked immunosorbent assay.
[0015] Furthermore, using the prepared polyclonal antibody against *Echinocactus glaucus* growth differentiation factor-8 / 11 as the primary antibody, and using *Echinocactus glaucus* coelomic fluid as the sample, the endogenous expression of *Echinocactus glaucus* growth differentiation factor-8 / 11 was analyzed by Western blotting. The main steps were as follows: *Echinocactus glaucus* coelomic fluid (with cell components removed) was concentrated and then separated by SDS-PAGE. The protein was transferred to a PVDF membrane by semi-dry electroporation, and after incubation with primary and secondary antibodies, substrate was added for development.
[0016] Compared with the prior art, the technical effects and advantages of the present invention are as follows: This invention yielded the *Echinochloa glabra* growth differentiation factor-8 / 11 antigen protein, a recombinant expression vector for the antigen protein, a polyclonal antibody, the full-length coding region of the *Echinochloa glabra* growth differentiation factor-8 / 11 gene, and primer pairs for amplifying this coding region. The preparation method provided by this invention enables the preparation of the *Echinochloa glabra* growth differentiation factor-8 / 11 antigen protein and polyclonal antibody, providing a valuable experimental tool for studying the function of *Echinochloa glabra* growth differentiation factor-8 / 11.
[0017] Specifically: (1) Providing a new antigen protein: This invention provides a novel growth differentiation factor-8 / 11 antigen protein of sea urchin glabra, whose amino acid sequence is shown in SEQ ID NO.1. This provides a new research object and material basis for related research fields and helps to explore the biological mechanisms related to the growth and differentiation of sea urchin glabra.
[0018] (2) Determining the full sequence of the gene coding region: The full sequence of the coding region of the growth differentiation factor-8 / 11 gene of sea urchin glabra was determined (as shown in SEQ ID NO.2). This sequence contains important structural information such as signal peptide sequence, TGF-β family domain and furin cleavage site, which provides key evidence for further research on the function, expression regulation and role of this gene in the growth and development of sea urchin glabra.
[0019] (3) Construction of a high-efficiency recombinant expression vector: A recombinant expression vector was constructed by inserting the coding sequence of the Echinococcus glabra growth differentiation factor-8 / 11 antigen protein into specific restriction enzyme sites (NdeI and XhoI) of the initial vector pET22b(+). This specific vector construction method is conducive to the high-efficiency expression of the target protein in Escherichia coli, providing a feasible and effective method for the large-scale preparation of Echinococcus glabra growth differentiation factor-8 / 11 antigen protein.
[0020] (4) Optimized antigen protein preparation method: This invention describes in detail the preparation method of the growth differentiation factor-8 / 11 antigen protein of Echinocactus glaucus, including extracting the coding sequence corresponding to the secretory mature protein from the full-length gene coding region, constructing an expression vector through seamless cloning, transforming it into E. coli and inducing expression, and then obtaining high-purity recombinant protein through a series of purification steps. This method has clear steps, is highly operable, and can obtain a high concentration (0.334 μg / μL) of recombinant protein, providing sufficient protein samples for subsequent related studies.
[0021] (5) Preparation of high-titer polyclonal antibodies: Mice were immunized with the recombinant protein prepared in this invention, and a polyclonal antibody with a titer as high as 1:25600 was successfully obtained for the growth differentiation factor-8 / 11 in Echinococcus glabra. The high-titer antibody provides a sensitive and reliable tool for subsequent detection of the expression of growth differentiation factor-8 / 11 in Echinococcus glabra, which helps to accurately analyze the expression of this factor in different tissues and physiological states of Echinococcus glabra.
[0022] (6) Detection of endogenous proteins: Using the prepared polyclonal antibody, the expression of endogenous growth differentiation factor-8 / 11 in the coelomic fluid of *Echinocactus glaucus* was successfully detected by Western blotting. The molecular weight of the detected positive band was consistent with that of the active secretory protein (disulfide-linked homodimer) of *Echinocactus glaucus* growth differentiation factor-8 / 11. This indicates that the polyclonal antibody prepared in this invention has high specificity and accuracy and can be used to detect *Echinocactus glaucus* growth differentiation factor-8 / 11 in actual samples, providing an effective detection method for studying the physiological function of this factor in *Echinocactus glaucus*.
[0023] (7) Provide amplification primer pairs: This invention provides primer pairs for amplifying the full sequence of the coding region of the growth differentiation factor-8 / 11 gene of sea urchin glabra (sequences shown in SEQ ID NO. 3-4). The primer pairs are reasonably designed and can specifically amplify the target gene sequence, providing a convenient tool for further research on the gene, such as gene cloning, expression analysis, mutation research, etc., and helping to promote the in-depth development of research related to growth differentiation factor-8 / 11 of sea urchin glabra. Attached Figure Description
[0024] Figure 1 This image shows the amino acid sequence alignment results of the maturation (secretory type) growth differentiation factor-8 / 11 of *Echinochloa glabra*, *Anemone rubrum*, and *Anemone stellaria*. At represents *Anemone rubrum*. Actinia tenebrosa (Sequence known), Nv represents star-shaped sea anemone Nematostella vectensis (Sequence known), Mn represents sea urchin with thorns. Mesocentrotus nudus ; Figure 2 SDS-PAGE analysis of the recombinant protein expression of growth differentiation factor-8 / 11 in sea urchin *Echinochloa glabra*. Figure 3 Antibody titer profile prepared for enzyme-linked immunosorbent assay (ELISA) analysis; Figure 4 A Western blot analysis of the endogenous expression of growth differentiation factor-8 / 11 protein in sea urchin *Echinochloa glabra*. Detailed Implementation
[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0026] Example 1: Amplification of the full-length coding region of the growth differentiation factor-8 / 11 gene in Echinocactus glaucus. Based on the base sequence information of the growth differentiation factor-8 / 11 gene transcript obtained from the transcriptome sequencing results of coelomic cells of *Echinocactus glaucus* obtained in previous experiments, primer pair ① was designed. Using cDNA obtained by reverse transcription of total RNA from coelomic cells of *Echinocactus glaucus* as a template, the target fragment was amplified by PCR, and a plasmid ligated with the target fragment was obtained by TA cloning and sequencing. A new full-length coding region sequence of the growth differentiation factor-8 / 11 gene of *Echinocactus glaucus* (1518 bp, sequence shown in SEQ ID NO.2) was obtained. The corresponding amino acid sequence contains a signal peptide sequence and a TGF-β family domain, as well as an RKRR sequence (i.e., the furin cleavage site). The sequence following the furin cleavage site is the inferred amino acid sequence of the mature secretory growth differentiation factor-8 / 11. This sequence was compared with that of red sea anemones (*Echinocactus rubescens*). Actinia tenebrosa ) and star-shaped sea anemones ( Nematostella vectensis The amino acid sequence alignment of known mature secretory growth differentiation factor-8 / 11 shows a consistent conserved cysteine structure. Figure 1 ).
[0027] Table 1 Primer Sequences Example 2: Preparation of growth differentiation factor-8 / 11 antigen protein from sea urchin *Echinochloa glabra* The coding sequence corresponding to the secretory mature protein of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene (underlined in SEQ ID NO.2) was extracted as the insertion sequence for constructing the recombinant antigen protein expression vector. Its corresponding amino acid sequence is shown in SEQ ID NO.1. Using the plasmid obtained by TA cloning in Example 1, which linked the entire coding sequence of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene, as a template, the coding sequence corresponding to the secretory mature protein of *Echinocactus glaucus* growth differentiation factor-8 / 11 was inserted between the NdeI and XhoI restriction sites of the pET22b(+) restriction enzymes to construct the expression vector pET22b(+)-MnGDF8 / 11. Primer pair ② used for seamless cloning is shown in Table 1. The expression vector pET22b(+)-MnGDF8 / 11 was transformed into *Escherichia coli* Origami(DE3) using the heat shock method, and positive clones were screened.
[0028] The expression of the recombinant protein Origami(DE3)-pET22b(+)-MnGDF8 / 11 was induced by IPTG, and the expression of the recombinant protein was analyzed by SDS-PAGE after bacterial cell lysis. The results are as follows: Figure 2As shown in the diagram (lane 1 is the molecular weight standard, lane 2 is the supernatant of the control group lysate, lane 3 is the precipitate of the control group lysate, lane 4 is the supernatant of the experimental group lysate, and lane 5 is the precipitate of the experimental group lysate), the expression of recombinant protein MnGDF8 / 11 was detected in the precipitate fraction of the induced bacterial cell lysate, suggesting that the recombinant protein is expressed in inclusion bodies. After nickel affinity chromatography, gradient dialysis, and concentration, the recombinant protein concentration was obtained as 0.334 μg / μl.
[0029] Example 3: Preparation of polyclonal antibody against growth differentiation factor-8 / 11 in sea urchin *Echinochloa glabra* The recombinant protein obtained in Example 2 was used as the antigen, and mice were immunized with an oil-in-water rapid adjuvant. The injection site was the calf muscle of the mouse's hind limb. A second immunization was performed 21 days after the initial immunization. Blood was collected from the eyeballs on day 35 after the initial immunization, serum was separated, and antibody titers were detected by indirect enzyme-linked immunosorbent assay (ELISA). The titer results are as follows: Figure 3 As shown, the valence is 1:25600.
[0030] Example 4: Using the prepared polyclonal antibody, the expression of endogenous growth differentiation factor-8 / 11 in the coelomic fluid of *Echinochloa glabra* was detected by Western blotting. Using a polyclonal antibody against *Echinocactus glaucus* growth differentiation factor-8 / 11 as the primary antibody, and *Echinocactus glaucus* coelomic fluid as the sample, the endogenous expression of *Echinocactus glaucus* growth differentiation factor-8 / 11 was analyzed by Western blotting. The main steps were as follows: *Echinocactus glaucus* coelomic fluid (after removing cellular components) was concentrated, and proteins were separated by SDS-PAGE. Proteins were then transferred to a PVDF membrane using a semi-dry electroporation method. After incubation with primary and secondary antibodies, substrate was added for development. Results are as follows: Figure 4 As shown, the experimental group lanes showed obvious positive bands between 25kD and 33kD, which are consistent with the molecular weight of the active secretory protein (disulfide-linked homodimer) of Echinococcus glabra growth differentiation factor-8 / 11, suggesting that the prepared polyclonal antibody against Echinococcus glabra growth differentiation factor-8 / 11 can be used for the detection of protein immunoblotting.
[0031] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A growth differentiation factor-8 / 11 antigen protein of *Echinocactus glaucus*, characterized in that, The amino acid sequence of the *Echinochloa glabra* growth differentiation factor-8 / 11 antigen protein is shown in SEQ ID NO.
1.
2. A complete sequence of the coding region of the growth differentiation factor-8 / 11 gene of *Echinochloa glabra*, characterized in that, The protein contains the coding sequence of the *Echinochloa glabra* growth differentiation factor-8 / 11 antigen protein as described in claim 1, wherein the nucleotide sequence of the coding region of the *Echinochloa glabra* growth differentiation factor-8 / 11 gene is shown in SEQ ID NO.
2.
3. A recombinant expression vector for the growth differentiation factor-8 / 11 antigen protein of *Echinochloa glabra*, characterized in that, It includes the coding sequence of the growth differentiation factor-8 / 11 antigen protein of sea urchin with Echinocactus glaucus as described in claim 2 and the initial vector.
4. The recombinant expression vector according to claim 3, characterized in that, The initial vector is pET22b(+), and the coding sequence of the Echinocactus glaber growth differentiation factor-8 / 11 antigen protein is inserted between the NdeI and XhoI restriction sites of the initial vector.
5. A method for preparing the growth differentiation factor-8 / 11 antigen protein of *Echinochloa glabra*, characterized in that, The method includes the following steps: transforming the recombinant expression vector of claim 3 or 4 into Escherichia coli, recombinantly expressing the target protein, and purifying it to obtain the recombinantly expressed Echinococcus lucida growth differentiation factor-8 / 11 antigen protein.
6. A polyclonal antibody against growth differentiation factor-8 / 11 in sea urchin *Echinochloa glabra*, characterized in that, The polyclonal antibody was obtained by immunizing mice with the Echinocactus glaucus growth differentiation factor-8 / 11 antigen protein as described in claim 1.
7. A primer pair for amplifying the full sequence of the coding region of the *Echinochloa glabra* growth differentiation factor-8 / 11 gene as described in claim 2, characterized in that, The sequences of the primer pairs are shown in SEQ ID NO.3-4.
8. The *Echinochloa glabra* growth differentiation factor-8 / 11 antigen protein according to claim 1, characterized in that, The amino acid sequence corresponding to the full-length coding region of the antigen protein contains a signal peptide sequence and a TGF-β family domain, as well as a furin cleavage site. The sequence following the furin cleavage site is the predicted mature secretory growth differentiation factor-8 / 11 amino acid sequence, which has a conserved cysteine structure consistent with the known mature secretory growth differentiation factor-8 / 11 amino acid sequences of red sea anemones and star-shaped sea anemones.
9. The method for preparing the *Echinochloa glabra* growth differentiation factor-8 / 11 antigen protein according to claim 5, characterized in that, The coding sequence corresponding to the secretory mature protein in the full-length coding region of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene as described in claim 2 was extracted as the insertion sequence for constructing the recombinant antigen protein expression vector. Using a plasmid obtained by TA cloning and linked to the full-length coding region of the *Echinocactus glaucus* growth differentiation factor-8 / 11 gene as a template, the coding sequence corresponding to the secretory mature protein of *Echinocactus glaucus* growth differentiation factor-8 / 11 was inserted between the NdeI and XhoI restriction sites of the pET22b(+) restriction enzyme in the vector to construct the expression vector pET22b(+)-MnGDF8 / 11. The primer pairs used for seamless cloning are shown in Table 1. The expression vector pET22b(+)-MnGDF8 / 11 was transformed into *E. coli* Origami(DE3) using a heat shock method, and positive clones were screened. The expression of the recombinant protein Origami(DE3)-pET22b(+)-MnGDF8 / 11 was induced by IPTG, and the cells were lysed and subjected to SDS-PAGE. PAGE analysis was performed to determine the expression of the recombinant protein. The recombinant protein was obtained by nickel affinity chromatography, gradient dialysis, and concentration.
10. A method for detecting the expression of endogenous growth differentiation factor-8 / 11 in the coelomic fluid of *Ulmus glabra* using the polyclonal antibody of *Ulmus glabra* as described in claim 6, characterized in that, Using the prepared polyclonal antibody against *Echinococcus opticii* growth differentiation factor-8 / 11 as the primary antibody, and using *Echinococcus opticii* coelomic fluid as the sample, the endogenous expression of *Echinococcus opticii* growth differentiation factor-8 / 11 was analyzed by Western blotting. The main steps were as follows: *Echinococcus opticii* coelomic fluid (after removing cellular components) was concentrated and then separated by SDS-PAGE. The protein was transferred to a PVDF membrane by semi-dry electroporation, and after incubation with primary and secondary antibodies, substrate was added for development.