Pseudosciaena crocea SOCS3 gene promoter sequence and application thereof

Abstract

The invention discloses a promoter sequence of a large yellow croaker SOCS3 gene. The promoter sequence is as shown in SEQ ID NO. 1. The recombinant plasmid pGL3-SOCS3 is used for transfecting a carp epithelioma (EPC) cell, renilla luciferase is used as an internal reference, and the activity of firefly luciferase can be 4.2 times of that of a control group at most, which shows that the large yellow croaker SOCS3 promoter promotes the transcription expression of a downstream reporter gene and has the basic activity of the promoter. Recombinant plasmids pGL3-SOCS3 and pRL-TK are co-transfected into EPC cells, then poly (I: C) is added, and the result shows that the poly (I: C) can significantly enhance the activity of the large yellow croaker SOCS3 promoter, and the activity of the large yellow croaker SOCS3 promoter is 1.7 times higher than that of a control group, indicating that the large yellow croaker SOCS3 promoter can respond to exogenous immunostimulation. The invention provides an excellent experimental system for studying the transcriptional regulation mechanism of the large yellow croaker SOCS3 gene, can also be used for efficiently expressing exogenous genes or studying transgenic large yellow croaker, and has theoretical significance and practical value.

Description

Technical Field

[0001] The present invention relates to the promoter of the large yellow croaker SOCS3 gene, in particular to the SOCS3 gene promoter sequence and its application. Background Art

[0002] Suppressor of cytokine signaling (SOCS) is a class of intracellular proteins that mainly act on the tyrosine kinase / signal transducer and activator of transcription (JAK-STAT) signaling pathway. By inhibiting the phosphorylation of STAT monomers, it inhibits the transmission of cytokine signals through the JAK-STAT signaling pathway (Starr R, Willson T A, Viney E M, et al. A family of cytokine-inducible inhibitors of signalling[J]. Nature, 1997, 387(6636):917-921.). SOCS3 is an inhibitor of multiple cytokines, most typically members of the gp130 family, including IL-6, IL-11, and LIF (Auernhammer C J, Bousquet C, Melmed S. Autoregulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter[J]. Proc Natl Acad Sci U SA, 1999, 96(12):6964-6969.). Herpes simplex virus (HSV) infection of human cells can stimulate the expression of SOCS3, thereby inhibiting the production of type I IFN (Michaud F, Coulombe F, Gaudreault E, et al. Epstein-Barr virus interferes with the amplification of IFNalpha secretion by activating suppressor of cytokine signaling 3 in primary human monocytes[J]. PLoS One, 2010, 5(7):e11908.). The sequences and functions of SOCS3 in fish have been reported. For example, overexpression of SOCS3 in turbot macrophages inhibited the transcription of TNF-α, IL-1β, and CC chemokines, and reduced the respiratory burst activity, nitric oxide production, and bactericidal activity of macrophages (Kiu H, Nicholson S E. Biology and significance of the JAK / STAT signalling pathways[J]. Growth Factors, 2012, 30(2):88-106.).Japanese flounder IL-6 induces the expression of SOCS3 and regulates PI3K-AKT-mediated signal transduction (Yoshimura A, Suzuki M, Sakaguchi R, et al. SOCS, Inflammation, and Autoimmunity [J]. Front Immunol, 2012, 3:20.). These results indicate that SOCS3 plays an important role in regulating the immune response of fish. At the same time, the expression of SOCS3 is strictly regulated at both the mRNA and protein levels, but there is no relevant report in fish. In the present invention, the promoter sequence of the SOCS3 gene of large yellow croaker was cloned. It was confirmed by the dual-luciferase reporter gene system analysis that this promoter sequence has promoter activity. Under the stimulation of the immune stimulant Poly(I:C), the promoter activity of the SOCS3 gene was significantly enhanced, indicating that the promoter sequence of the SOCS3 gene of large yellow croaker and its recombinant plasmid can provide an experimental system for studying the transcriptional regulation mechanism of the SOCS3 gene, and can also be used for the efficient expression of foreign genes or the research of transgenic fish, which has important theoretical and practical significance. Summary of the Invention

[0003] One object of the present invention is to provide the sequence of the promoter of the SOCS3 gene of large yellow croaker.

[0004] Another object of the present invention is to provide a method for analyzing the promoter activity of the SOCS3 gene of large yellow croaker.

[0005] A further object of the present invention is to provide an application of the promoter of the SOCS3 gene of large yellow croaker.

[0006] The sequence of the promoter of the SOCS3 gene of large yellow croaker described in the present invention is shown in SEQ ID NO.1.

[0007] The method for analyzing the promoter activity of the SOCS3 gene of large yellow croaker described in the present invention is as follows:

[0008] Using the genomic DNA of large yellow croaker as a template, the promoter sequence of the SOCS3 gene of large yellow croaker was amplified by PCR and ligated to the luciferase reporter gene vector pGL3-Basic plasmid of Promega Corporation to obtain the pGL3-SOCS3 recombinant plasmid. The recombinant plasmid pGL3-SOCS3 and the control plasmid pGL3-Basic were respectively transfected into epithelioma papulosum cyprini (EPC) cells. Using the Renilla luciferase reporter gene plasmid pRL-TK as an internal reference, the dual-luciferase reporter gene detection system was used to analyze the difference in firefly luciferase activity between different groups. Then, the EPC cells transfected with the pGL3-SOCS3 plasmid were stimulated with Poly(I:C) to analyze the change in firefly luciferase activity.

[0009] The application of the large yellow croaker SOCS3 gene promoter is as follows:

[0010] The promoter sequence of the large yellow croaker SOCS3 gene provided by the present invention has promoter basal activity, can initiate the transcription and expression of downstream reporter genes, and respond to Poly(I:C) stimulation, providing an excellent experimental system for studying the transcriptional regulation mechanism of the large yellow croaker SOCS3 gene, and can also be used for highly expressing foreign genes or applied to the research of transgenic large yellow croakers, having theoretical significance and practical value.

[0011] The beneficial effect of the present invention is that: the present invention provides a promoter sequence of the large yellow croaker SOCS3 gene. The recombinant plasmid pGL3-SOCS3 and the control plasmid pGL3-Basic are respectively transfected into epithelioma papulosum cyprini (EPC) cells. Using the Renilla luciferase reporter gene plasmid pRL-TK as an internal reference, the dual-luciferase reporter gene detection system is used to analyze the differences in firefly luciferase activity between different groups. The results show that the firefly luciferase activity in EPC cells transfected with the pGL3-SOCS3 recombinant plasmid is significantly higher than that in the control group, up to 4.2 times that of the control group at most, indicating that the large yellow croaker SOCS3 promoter initiates the transcription and expression of downstream reporter genes and has promoter basal activity.

[0012] In the present invention, the recombinant plasmids pGL3-SOCS3 and pRL-TK are co-transfected into EPC cells, and then poly(I:C) is added. The results show that Poly(I:C) can significantly enhance the activity of the large yellow croaker SOCS3 promoter, rising by up to 1.7 times compared with the control group, indicating that the large yellow croaker SOCS3 promoter can respond to exogenous immune stimulation. Description of the Drawings

[0013] Figure 1 To analyze the activity of the large yellow croaker SOCS3 gene promoter using the dual-luciferase reporter gene detection system; the experimental group is transfected with the pGL3-SOCS3 recombinant plasmid; the control group is transfected with the pGL3-Basic plasmid.

[0014] Figure 2 For the change in the activity of the large yellow croaker SOCS3 gene promoter after Poly(I:C) stimulation. Detailed Embodiments

[0015] The following examples will further illustrate the present invention in conjunction with the drawings.

[0016] Example 1 Cloning of the Large Yellow Croaker SOCS3 Gene Promoter

[0017] The genomic DNA of large yellow croaker gill tissue was extracted using the Marine Animal Tissue Genomic DNA Extraction Kit (Tiangen, Beijing). Using the genomic DNA as a template, the promoter sequence of the large yellow croaker SOCS3 gene was amplified by PCR technology, ligated to the luciferase reporter plasmid pGL3-Basic of Promega Corporation, transformed into Escherichia coli DH5α competent cells, and positive clones were screened by colony PCR and sequencing to obtain the pGL3-SOCS3 recombinant plasmid. The PCR primers used were:

[0018] Forward primer: 5'-TCTATCGATAGGTACTGCCAAAAAAAAACTATCCATTA-3';

[0019] Reverse primer: 5'-GATCTCGAGCCCGGGACAAACTTCAGACTTGTTAAACACTT-3'.

[0020] Example 2 Analysis of the promoter activity of the large yellow croaker SOCS3 gene

[0021] EPC cells were inoculated into 96-well plates and cultured overnight at 28°C. The cell culture medium was M199 medium from Boshark Biotech. The plasmid was transfected into EPC cells using the Lipo8000 TM transfection reagent from Beyotime. The transfection complex was prepared according to 1 μg plasmid and 2 μL transfection reagent (mass-volume ratio of 1:2), added to the serum-free medium, and the EPC cells were incubated and cultured. The experimental group was co-transfected with the pGL3-SOCS3 recombinant plasmid and the internal reference pRL-TK plasmid (100:1) into EPC cells; the control group was co-transfected with the pGL3-Basic plasmid and the internal reference pRL-TK plasmid (100:1) into EPC cells. Each experiment was set with three replicates, and each replicate was set with three parallels. Cells were collected at 18 h, 24 h, 36 h, and 48 h after transfection, and the change in the activity of firefly luciferase in the cells was detected using the Dual-Luciferase Reporter Assay System from Promega Corporation and a microplate multimode luminometer Discover Microplate Reader according to the instructions. The results showed that the activity of firefly luciferase in EPC cells transfected with the pGL3-SOCS3 recombinant plasmid was significantly higher than that of the control group, up to 4.2 times that of the control group Figure 1 ), indicating that the large yellow croaker SOCS3 promoter initiated the transcriptional expression of the downstream reporter gene and had promoter basal activity.

[0022] Example 3 Activation effect of immune stimulants on the large yellow croaker SOCS3 promoter

[0023] The recombinant plasmids pGL3-SOCS3 and pRL-TK were co-transfected into EPC cells. After culturing for 12 h, poly(I:C) with a final concentration of 10 μg / mL was added, and PBS was used as a control. After continuing to culture for 12 h, 24 h, and 36 h, the changes in the activity of firefly luciferase in the cells were analyzed according to the above experimental procedures. Each experiment was set with three replicates, and each replicate was set with three parallels. The results showed that Poly(I:C) could significantly enhance the activity of the large yellow croaker SOCS3 promoter, which increased by up to 1.7 times compared with the control group, indicating that the large yellow croaker SOCS3 promoter could respond to exogenous immune stimulation( Figure 2 ).

[0024] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A large yellow croaker SOCS3 gene promoter, characterized in that: The promoter sequence is shown as SEQ ID NO.

1.

2. A recombinant plasmid pGL3-SOCS3, characterized in that: The method comprises the promoter according to claim 1 and a luciferase reporter gene.

3. The recombinant plasmid pGL3-SOCS3 according to claim 2, characterized in that: The recombinant plasmid is obtained by connecting the large yellow croaker SOCS3 gene promoter sequence to the luciferase reporter gene vector pGL3-Basic plasmid of Promega Company.

4. A method for analyzing the activity of the SOCS3 gene promoter of large yellow croaker as claimed in claim 1, characterized in that: The recombinant plasmid pGL3-SOCS3 and the control plasmid pGL3-Basic were transfected into carp epithelial tumor cells EPC, and the Renilla luciferase reporter gene plasmid pRL-TK was used as an internal reference. The dual luciferase reporter gene detection system was used to analyze the differences in firefly luciferase activity among different groups. Poly(I:C) was then used to stimulate EPC cells transfected with pGL3-SOCS3 plasmid, and the changes in firefly luciferase activity were analyzed.

5. An application of the large yellow croaker SOCS3 gene promoter as claimed in claim 1, characterized in that: These include: (1) To study the transcriptional regulation mechanism of SOCS3 gene in large yellow croaker; (2) Efficient expression of exogenous genes; (3) Research on genetically modified large yellow croaker.

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