Pseudosciaena crocea interleukin 4 / 13B gene promoter sequence and application thereof

Abstract

The invention discloses a promoter of a larimichthys crocea interleukin 4 / 13B (IL-4 / 13B) gene. The sequence of the promoter is as shown in SEQ ID NO. 1. A recombinant plasmid pGL3-IL-4 / 13B and a control plasmid pGL3-Basic are used for respectively transfecting carp epithelioma EPC cells, a renilla luciferase reporter gene plasmid pRL-TK is used as an internal reference, and a dual-luciferase reporter gene detection system is used for analyzing the activity difference of firefly luciferase among different groups; and then, respectively using Poly (I: C), phytolectin PHA and inactivated pseudomonas protegens to stimulate EPC cells transfected with pGL3-IL-4 / 13B plasmids, and analyzing the change of the activity of firefly luciferase. The promoter sequence of the large yellow croaker IL-4 / 13B gene provided by the invention has the basic activity of the promoter, can start the transcription expression of a downstream reporter gene, and responds to the exogenous stimulation of Poly (I: C), PHA, inactivated pseudomonas proteinans and the like, so that an excellent experimental system is provided for researching the transcription regulation mechanism of the large yellow croaker IL-4 / 13B gene; the method can also be used for efficiently expressing exogenous genes or researching transgenic pseudosciaena crocea, and has theoretical significance and practical value.

Description

Technical Field

[0001] The present invention relates to a promoter of the large yellow croaker interleukin-4 / 13B (IL-4 / 13B) gene, and particularly relates to an IL-4 / 13B gene promoter sequence and its application. Background Art

[0002] Interleukin-4 (IL-4) and IL-13 are cytokines with similar structures and functions, mainly produced by Th2 cells (T-helper cell type 2), and mediate humoral immune responses by promoting Th cell differentiation, inducing major histocompatibility complex (MHC) II protein expression, antibody production, antibody class switching, etc. (McCormick M S, Heller M N. Commentary: IL-4 and IL-13 receptors and signaling [J]. Cytokine, 2015, 75(1): 38-50.). Fish IL-4 / 13 is a homologous gene of mammalian IL-4 and IL-13. The homologous gene close to RAD50 on the genome is named IL-4 / 13A, and the gene close to KIF3A is named IL-4 / 13B. Fish IL-4 / 13 can promote B cell proliferation, differentiation, and antibody secretion (Roy B, Bhattacharjee A, Xu B, et al. IL-13 signal transduction in human monocytes: phosphorylation of receptor components, association with Jaks, and phosphorylation / activation of Stats [J]. J Leukoc Biol. 2002, 72(3): 580-589).

[0003] Large yellow croaker IL-4 / 13B is a pleiotropic cytokine, which has been proven to significantly inhibit the production of superoxide anions, reactive oxygen species and nitric oxide in monocytes / macrophages, down-regulate the expression of inflammatory factors in monocytes / macrophages, and promote the expression of growth-related factors, thus promoting the activation of monocytes / macrophages towards the M2 type (Mao K, Chen W, Mu Y, et al. Identification of two IL-4 / 13 homologues in large yellow croaker (Larimichthys crocea) revealed their similar roles in inducing alternative activation of monocytes / macrophages [J]. Fish and Shellfish Immunology, 2018, 80: 180-190). In the present invention, the promoter sequence of the large yellow croaker IL-4 / 13B gene was cloned. Analysis by the dual-luciferase reporter gene system confirmed that this promoter sequence has promoter activity. Under the stimulation of immune stimulants Poly(I:C), PHA and inactivated Pseudomonas plecoglossicida, the promoter activity of the IL-4 / 13B gene was significantly enhanced, indicating that the promoter sequence of the large yellow croaker IL-4 / 13B and its recombinant plasmid can provide an experimental system for studying the transcriptional regulation mechanism of the IL-4 / 13B gene, and can also be used for the high-efficiency expression of foreign genes or the research of transgenic fish, which has important theoretical and practical significance. Summary of the Invention

[0004] One of the objectives of the present invention is to provide the sequence of the promoter of the large yellow croaker IL-4 / 13B gene.

[0005] Another objective of the present invention is to provide a method for analyzing the activity of the promoter of the large yellow croaker IL-4 / 13B gene.

[0006] A further objective of the present invention is to provide an application of the promoter of the large yellow croaker IL-4 / 13B gene.

[0007] The sequence of the promoter of the large yellow croaker IL-4 / 13B gene described in the present invention is shown as SEQ ID NO.1.

[0008] The method for analyzing the activity of the promoter of the large yellow croaker IL-4 / 13B gene described in the present invention is as follows:

[0009] Using the genomic DNA of large yellow croaker as a template, the promoter sequence of the large yellow croaker IL-4 / 13B gene was amplified by PCR and ligated to the luciferase reporter gene vector pGL3-Basic plasmid of Promega Corporation to obtain the recombinant plasmid pGL3-IL-4 / 13B. The recombinant plasmid pGL3-IL-4 / 13B 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 differences in firefly luciferase activity among different groups. Then, the EPC cells transfected with the pGL3-IL-4 / 13B plasmid were stimulated with Poly(I:C), phytohemagglutinin (PHA), and inactivated Pseudomonas plecoglossicida respectively, and the changes in firefly luciferase activity were analyzed.

[0010] The application of the large yellow croaker IL-4 / 13B gene promoter is as follows:

[0011] The promoter sequence of the large yellow croaker IL-4 / 13B gene provided by the present invention has promoter basal activity, can initiate the transcription and expression of downstream reporter genes, and respond to exogenous stimuli such as Poly(I:C), PHA, and inactivated Pseudomonas plecoglossicida, providing an excellent experimental system for studying the transcriptional regulation mechanism of the large yellow croaker IL-4 / 13B gene, and can also be used for highly expressing exogenous genes or applied to the research of transgenic large yellow croaker, having theoretical significance and practical value.

[0012] The beneficial effects of the present invention are as follows: The present invention discloses a promoter of the large yellow croaker interleukin 4 / 13B (IL-4 / 13B) gene, and the promoter sequence is as shown in SEQ ID NO.1. This promoter sequence has promoter basal activity, can initiate the transcription and expression of downstream reporter genes, and respond to exogenous stimuli such as Poly(I:C), PHA, and inactivated Pseudomonas plecoglossicida, providing an excellent experimental system for studying the transcriptional regulation mechanism of the large yellow croaker IL-4 / 13B gene, and can also be used for highly expressing exogenous genes or applied to the research of transgenic large yellow croaker, having theoretical significance and practical value.

[0013] In this invention, the pGL3-IL-4 / 13B recombinant plasmid and the internal reference pRL-TK plasmid were co-transfected into EPC cells. After 48 hours of transfection, it was measured that the luciferase activity in the EPC cells transfected with the pGL3-IL-4 / 13B recombinant plasmid was significantly higher than that of the control group, being 7.39 times that of the control group, indicating that the large yellow croaker IL-4 / 13B promoter initiated the transcriptional expression of the downstream reporter gene and had promoter basal activity. Additionally, stimulation with Poly(I:C), PHA, and heat-inactivated Pseudomonas plecoglossicida could significantly enhance the activity of the large yellow croaker IL-4 / 13B promoter, with 3.4-fold, 3.3-fold, and 1.5-fold upregulation respectively compared with the control group, indicating that the large yellow croaker IL-4 / 13B promoter could respond to exogenous immune stimulation. Brief Description of the Drawings

[0014] Figure 1 To analyze the activity of the large yellow croaker IL-4 / 13B gene promoter using the dual-luciferase reporter gene detection system; the experimental group was transfected with the pGL3-IL-4 / 13B recombinant plasmid; the control group was transfected with the pGL3-Basic plasmid.

[0015] Figure 2 For the changes in the activity of the large yellow croaker IL-4 / 13B gene promoter after different immune stimulations. Detailed Embodiments

[0016] The following embodiments will further illustrate the present invention in conjunction with the drawings.

[0017] Example 1 Cloning of the Large Yellow Croaker IL-4 / 13B Gene Promoter

[0018] The genomic DNA of large yellow croaker muscle tissue was extracted using a marine animal tissue genomic DNA extraction kit (Tiangen, Beijing). Using the genomic DNA as a template, the promoter sequence of the large yellow croaker IL-4 / 13B gene was amplified by PCR technology, ligated to the luciferase reporter plasmid pGL3-Basic of Promega Corporation, and transformed into Escherichia coli DH5α competent cells. Positive clones were screened by colony PCR and sequencing to obtain the pGL3-IL-4 / 13B recombinant plasmid. The PCR primers used were:

[0019] Forward primer 5’-TCTATCGATAGGTACAAGGTGACAGACTGTGGACTG-3',

[0020] Reverse primer: 5'-GATCTCGAGCCCGGGGTCTTTTGAAAGAGACCGCGAG-3'.

[0021] Example 2 Analysis of the Activity of the Large Yellow Croaker IL-4 / 13B Gene Promoter

[0022] EPC cells were seeded into 96-well plates and cultured overnight at 28°C. The cell culture medium was L-15 medium from Gibco. Plasmids were transfected into EPC cells using Fugene HD transfection reagent (Promega). Transfection complexes were prepared according to 0.1 μg plasmid and 0.2 μL transfection reagent (mass-volume ratio of 1:2) and added to serum-free medium to incubate and culture EPC cells. The experimental group was co-transfected with pGL3-IL-4 / 13B recombinant plasmid and internal reference pRL-TK plasmid (200:1) into EPC cells; the control group was co-transfected with pGL3-Basic plasmid and internal reference pRL-TK plasmid (200:1) into EPC cells. Each experiment was set with three replicates, and each replicate was set with three parallels. ** represents P < 0.01. After 48 h of transfection, the dual-luciferase reporter gene detection system from Promega and a microplate multimode luminometer ( Discover Microplate Reader) were used to analyze the changes in firefly luciferase activity in cells according to the instructions. The results showed ( Figure 1 ): The firefly luciferase activity in EPC cells transfected with pGL3-IL-4 / 13B recombinant plasmid was significantly higher than that in the control group, which was 7.39 times that of the control group, indicating that the large yellow croaker IL-4 / 13B promoter initiated the transcriptional expression of the downstream reporter gene and had promoter basal activity.

[0023] Example 3 Activation effect of immune stimulants on the large yellow croaker IL-4 / 13B promoter

[0024] The recombinant plasmids pGL3-IL-4 / 13B and pRL-TK were transfected into EPC cells. After 6 h of culture, poly(I:C), PHA, or inactivated Pseudomonas plecoglossicida (P.p) with a final concentration of 10 μg / mL were added to stimulate the cells. Using PBS as a control, after continuing to culture for 18 h, the changes in firefly luciferase activity in cells were analyzed according to the above experimental steps. Each experiment was set with three replicates, and each replicate was set with three parallels. ** represents P < 0.01. The results showed ( Figure 2 ): Stimulation with Poly(I:C), PHA, and inactivated Pseudomonas plecoglossicida could significantly enhance the activity of the large yellow croaker IL-4 / 13B promoter, which were upregulated by 3.4 times, 3.3 times, and 1.5 times respectively compared with the control group, indicating that the large yellow croaker IL-4 / 13B promoter could respond to exogenous immune stimuli.

[0025] 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 covered by the present invention.

Claims

1. A large yellow croaker interleukin 4 / 13B gene promoter, characterized in that: The promoter sequence is shown as SEQ ID NO.

1.

2. A recombinant plasmid pGL3-IL-4 / 13B, characterized in that: The recombinant plasmid comprises the large yellow croaker interleukin 4 / 13B gene promoter and the luciferase reporter gene as described in claim 1.

3. The recombinant plasmid pGL3-IL-4 / 13B according to claim 2, characterized in that: The recombinant plasmid is obtained by connecting the large yellow croaker IL-4 / 13B gene promoter sequence to the luciferase reporter gene vector pGL3-Basic plasmid.

4. A method for analyzing the activity of the interleukin 4 / 13B gene promoter of large yellow croaker as claimed in claim 1, characterized in that: The recombinant plasmid pGL3-IL-4 / 13B and the control plasmid pGL3-Basic were transfected into carp epithelial tumor EPC cells, respectively. The Renilla luciferase reporter gene plasmid pRL-TK was used as an internal reference, and the dual-luciferase reporter gene detection system was used to analyze the differences in firefly luciferase activity among different groups. Poly(I:C), plant lectin PHA and inactivated Pseudomonas aeruginosa were used to stimulate the EPC cells transfected with pGL3-IL-4 / 13B plasmid, and the changes in firefly luciferase activity were analyzed.

5. An application of the large yellow croaker interleukin 4 / 13B gene promoter as claimed in claim 1, characterized in that: These include: (1) As an experimental system for studying the transcriptional regulation mechanism of IL-4 / 13B gene in large yellow croaker; (2) Efficient expression of exogenous genes; (3) Research on genetically modified large yellow croaker.

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