Recombinant plasmid as well as preparation method and application thereof

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a recombinant plasmid as well as a preparation method and application thereof. According to the method, the CDS region of the DrTRIM13 gene of the zebra fish is amplified by adopting an RT-PCR technology, and the size of an amplified product is consistent with an expected result. According to the invention, a recombinant plasmid pEGFP-DrTRIM13 is also constructed, and after the recombinant plasmid pEGFP-DrTRIM13 is subjected to double enzyme digestion by using Hind-III and EcoR-I, the DrTRIM13 gene is connected into the plasmid pEGFP-N2. The sequencing result of the positive clone shows that the DrTRIM13 gene connected into the pEGFP-N2 vector is consistent with the base sequence obtained by sequencing, which indicates that the recombinant plasmid pEGFP-DrTRIM13 is successfully constructed.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and specifically relates to a recombinant plasmid and a preparation method and application thereof. Background Art

[0002] E3 ubiquitin ligases play important roles in innate immunity of teleosts. TRIM13 has ubiquitin ligase E3 activity and has been shown to play a key regulatory role in innate immunity mediated by TLR signaling. In mammals, TRIM proteins participate in antiviral immunity through a variety of mechanisms (such as TNFα, IFN-β, and IL-6), ranging from direct viral restriction to regulation of immune signaling and, most recently, autophagy. The molecular functions of mammalian TRIM13 proteins have been explored in multiple response pathways, but their role in fish innate immunity and inflammatory signaling remains understudied. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a recombinant plasmid, which is constructed by introducing the zebrafish DrTRIM13 gene into the multiple cloning site region of the PMD20-T vector.

[0004] The present invention also provides a method for preparing the above-mentioned recombinant plasmid, which can be prepared by using cDNA obtained by reverse transcription of zebrafish liver tissue as a template to PCR amplify the full-length sequence of the CDS region of DrTRIM13, and connecting the PCR product to the PMD20-T vector.

[0005] Furthermore, the amplification system of the full-length sequence of the DrTRIM13 CDS region is: 21 μL dd H2O, 1.0 μL of upstream and downstream primers, 25 μL 2×dNTP Mixture, 2 μL DNA template; the concentration of the upstream and downstream primers is 10 μmol / L.

[0006] Furthermore, the amplification conditions of the full-length sequence of the DrTRIM13 CDS region are: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, and 35 cycles; extension at 72°C for 10 min, and termination at 16°C.

[0007] The present invention also provides the use of the recombinant plasmid in preparing immune response products.

[0008] The present invention has the following beneficial effects:

[0009] The present invention cloned the TRIM13 homolog of zebrafish, clarified the gene expression in different tissues, performed bioinformatics analysis, and finally performed subcellular localization. The technology provided by the present invention helps to understand the function of TRIM gene in fish immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 The expression of DrTRIM13 gene and β-actin gene in various tissues; lanes 1-7 are intestine, kidney, muscle, gill, skin, liver and swim bladder, respectively;

[0012] Figure 2 M is a 2kb DNA Marker, and 1 is the double enzyme digestion product of the recombinant plasmid pEGFP-DrTRIM13.

[0013] Figure 3 Subcellular localization of DrTRIM1 in HEK293T cells (200×) DETAILED DESCRIPTION

[0014] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0015] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0016] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0017] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0018] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0019] Example

[0020] 1. Experimental Materials

[0021] Zebrafish were purchased from Fengqi Flower and Bird Market, Hangzhou, China. The temperature of the breeding pond was maintained at 28±1℃. After 1 week of breeding, the liver, intestine, muscle, skin, swim bladder, gills and kidney were dissected and separated. They were immediately treated with liquid nitrogen and stored at -80℃ for later use. 2×TaqPCR Mix, PrimeScriptTM IV 1st strand cDNA Synthesis Mix kit, reverse transcription kit, restriction endonuclease, DNA marker, RNAiso Plus, pMD 20 and T4 ligase were purchased from Takara, and DNA purification and recovery kit and plasmid mini-preparation kit were purchased from OMEGA. STBL3 competent cells, pEGFP-N2 vector and HEK 293T cell line were stored in this laboratory. DMEM high glucose medium, phosphate buffered saline (PBS) and fetal bovine serum were purchased from Gibco, and trypsin was purchased from Shanghai Jitai Biotechnology Co., Ltd. Cationic liposome Lipofectamine 2000 was purchased from Invitrogen.

[0022] 2.2 RNA extraction and cDNA synthesis

[0023] According to the experimental instructions, RNAiso Plus reagent was used to extract total RNA from various tissues of zebrafish. 1 μg of total RNA was used for reverse transcription reaction, and cDNA was obtained by reverse transcription using PrimeScriptTM kit and stored at -20℃ for future use.

[0024] 2.3 Cloning of the full-length zebrafish DrTRIM13 CDS

[0025] According to the sequence of zebrafish TRIM13 (sequence number: NM_001020603.1) in NCBI, Prime 5.0 software was used to design a pair of primers DrTRIM13-F / DrTRIM13-R (Table 1). The cDNA obtained by reverse transcription from the liver was used as a template to amplify the full-length sequence of the CDS region of DrTRIM13. PCR amplification system (50μL): 21μL dd H2O, 1.0μL each of the upstream and downstream primers (10μmol / L), 25μL 2×dNTPMixture, 2μL DNA template. PCR amplification conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 60s, 35 cycles; 72℃ extension for 10min, 16℃ termination.

[0026] The PCR product was detected by 1.2% agarose gel electrophoresis and recovered from the gel, then connected to the PMD20-T vector and transformed into STBL 3 competent cells, positive clones were screened and plasmids were extracted, and sequenced by Shanghai Shenggong Biotechnology Co., Ltd. Finally, the sequence was spliced ​​using DNA Star software to obtain the cDNA sequence of the CDS region of the DrTRIM13 gene.

[0027] Table 1 Primers and primer sequences

[0028]

[0029] 2.4 Zebrafish tissue expression profiling analysis

[0030] The expression of zebrafish DrTRIM13 gene in skin, muscle, liver, intestine, swim bladder, gill and kidney was studied by RT-PCR. β-actin was used as the internal reference gene. A pair of primers β-actin-F / β-actin-R (Table 1) were designed according to the CDS sequence of zebrafish β-actin (sequence number: NM_001020603.1) in NCBI. The primers and reaction system used for PCR refer to 2.3, the annealing temperature is 59 ° C, and the annealing time is 30s. The PCR product was then electrophoresed in 1.2% agarose gel for analysis of PCR results.

[0031] 2.5 Construction and identification of recombinant plasmid pEGFP-DrTRIM13

[0032] According to the deduced sequence fragment of zebrafish TRIM13 gene, a pair of primers DrTRIM13-Hind-III-F / DrTRIM13-EcoR-Ⅰ-R was designed, and the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The plasmid obtained in 2.3 was used as a template, and the PCR amplification system was referred to 2.3. In order to determine the function of TRIM13 gene in vitro, the gel-recovered product of PCR and the pEGFP-N2 vector were double-digested with Hind-III and EcoR-Ⅰ, and then the two target fragments were connected at 16℃ for 2-4h with T4 ligase, and the ligation product was transformed into STBL 3 competent cells to construct pEGFP-DrTRIM13 plasmid. The above transformation products were plated on LB solid medium containing kanamycin (Kana), and the transformed colonies were screened. The single colony was picked every other day, and the plasmid was extracted, identified by double enzyme digestion, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing identification.

[0033] 2.6 Subcellular localization of zebrafish DrTRIM13 gene

[0034] The successfully constructed expression plasmid pEGFP-DrTRIM13 was transfected into human embryonic kidney HEK293T cells for subcellular localization. HEK293T cells were cultured with DMEM (containing 10% fetal bovine serum) at 37°C and 5% CO2, and fresh culture medium was replaced every 2-3 days. When the cell confluence rate reached about 90%, 0.25% trypsin was used for digestion for 2-3 minutes, fresh culture medium was added to terminate the digestion, and the cells were diluted 1:4, and repeatedly blown and mixed to make a single cell suspension. 20μL of single cell suspension was added to the center of the coverslip placed in the 6-well plate for cell climbing culture. After 24 hours, the fluorescent fusion expression plasmid pEGFP-TRIM13 was transfected into HEK293T cells with Lipofectamine 2000 transfection reagent. The empty pEGFP-N2 plasmid was set as a negative control and transfected at the same time. The medium was changed 4-6 hours after transfection, and the culture medium was discarded 48 hours later. The slides were fixed with formalin for 10 minutes, washed twice with PBS, and finally stained with 0.1 μg / mL LDAPI for 2 minutes. After discarding, the slides were washed with PBS and then sealed with 30% glycerol. Fluorescence photography was performed with an Olymapus upright microscope.

[0035] Results Analysis

[0036] Figure 1 The results showed that the DrTRIM13 gene was widely expressed in adult zebrafish tissues such as skin, muscle, liver, intestine, swim bladder, gills, and kidneys.

[0037] Figure 2 The results showed that there was a cut band between 1000 bp and 2000 bp, indicating that the DrTRIM13 gene had been linked to the pEGFP-N2 plasmid.

[0038] in conclusion

[0039] 1. Amplification results of the CDS region of zebrafish DrTRIM13 gene

[0040] In this study, RT-PCR technology was used to amplify the CDS region of the zebrafish DrTRIM13 gene, and the size of the amplified product was consistent with the expected result.

[0041] 2 Double restriction enzyme digestion identification of recombinant plasmid pEGFP-DrTRIM13

[0042] After the recombinant plasmid pEGFP-DrTRIM13 was double-digested with Hind-III and EcoR-Ⅰ, the DrTRIM13 gene was linked into the pEGFP-N2 plasmid. The sequencing results of the positive clones showed that the DrTRIM13 gene linked into the pEGFP-N2 vector was consistent with the base sequence obtained by sequencing, indicating that the recombinant plasmid pEGFP-DrTRIM13 was successfully constructed.

[0043] Subcellular localization of 3DrTRIM13

[0044] The expression and localization results of DrTRIM13 protein in cells in vitro showed that the green fluorescence of the control pEGFP-N2 transfected cells was evenly distributed throughout the cells, while the green fluorescence of pEGFP-DrTRIM13 was mainly distributed in the cytoplasm of HEK293T cells, occasionally aggregated into round dots.

[0045] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant plasmid, characterized in that: The recombinant plasmid is constructed by introducing the zebrafish DrTRIM13 gene into the multiple cloning site region of the PMD20-T vector.

2. The method for preparing a recombinant plasmid according to claim 1, characterized in that: The full-length sequence of the CDS region of DrTRIM13 was amplified by PCR using the cDNA obtained by reverse transcription from zebrafish liver tissue as a template, and the PCR product was connected to the PMD20-T vector to obtain the obtained product.

3. The preparation method according to claim 2, characterized in that: The amplification system of the full-length sequence of the DrTRIM13 CDS region is: 21 μL dd H2O, 1.0 μL of upstream and downstream primers, 25 μL 2×dNTP Mixture, and 2 μL DNA template; the concentration of the upstream and downstream primers is 10 μmol / L.

4. The preparation method according to claim 2, characterized in that: The amplification conditions of the full-length sequence of the DrTRIM13 CDS region are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for 10 min, and termination at 16°C.

5. Use of the recombinant plasmid as claimed in claim 1 in preparing an immune response product.

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