Construction and application of over-expression vectors of three subtypes of porcine PDLIM3 protein

A sequence listing and technology in the sequence listing, applied in the field of construction of overexpression vectors of three different subtypes of porcine PDLIM3 protein, can solve problems such as influence and change of function, and achieve enhanced expression, high-efficiency expression, simple and feasible construction method

Inactive Publication Date: 2015-06-10
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, a large number of alternative splicing phenomena have been found in the 3'UTR, which is likely to change its RNA secondary structure, affect its transcription and thus change its function

Method used

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  • Construction and application of over-expression vectors of three subtypes of porcine PDLIM3 protein
  • Construction and application of over-expression vectors of three subtypes of porcine PDLIM3 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Amplification of three different subtype gene sequences of porcine PDLIM3 protein

[0015] (1) Extraction of total RNA

[0016] 1. Put 100mg of muscle tissue into a mortar filled with liquid nitrogen, and grind the tissue into powder with a pestle.

[0017] 2. Transfer the powder to an Eppendorf tube, add 1.0 mL of Trizol per 100 mg of tissue, grind thoroughly with a homogenizer, and transfer to a centrifuge tube.

[0018] 3. Centrifuge at 12000r / min for 10min at 4°C to separate the mixture into two phases.

[0019] 4. Transfer the upper aqueous phase to another centrifuge tube, add 200μL chloroform / 1mL Trizol, mix thoroughly, let stand at room temperature for 5 minutes, and centrifuge at 12000r / min for 10min at 4°C.

[0020] 5. Carefully transfer the supernatant to a 1.5mL centrifuge tube, add an equal volume of isopropanol to the supernatant, and place at room temperature for 15 minutes.

[0021] 6. Centrifuge at 12000r / min for 10min to precipitate RNA. ...

Embodiment 2

[0033] Example 2: Construction of different PDLIM3 subcellular localization vectors

[0034] (1) Amplification of different transcripts of porcine PDLIM3 The amplified products were purified with an agarose gel purification kit.

[0035] (2) Prepare the pEGFP-N1 empty vector by extraction with a plasmid mini-extraction kit.

[0036] (3) The purified PCR product and the extracted plasmid were digested with EcoR1 and BamH1 restriction endonucleases respectively, and purified and recovered after incubation at 37°C for 6 hours. Double enzyme digestion system for PCR product and vector plasmid: the total volume is 20 μL, including 8 μL of pEGFP-N1 vector DNA, 2 μL of 10×Buffer (MBI), 1 μL of EcoR1 and BamH1 restriction enzymes, double Distilled water 8 μL.

[0037] (4) Use ligase T4 DNA Ligase for enzyme ligation. The enzyme ligation system is: 2 μL of plasmid double digestion product, 6 μL of PDLIM3 gene fragment double digestion product, T4 Ligase 1 μL, 10×Buffer 1 μL, after mi...

Embodiment 3

[0039] Example 3: Subcellular localization of three different isoforms of porcine PDLIM3 protein

[0040] (1) Cell culture

[0041] Porcine kidney cells (Porcine kidney cells, PK15) were cultured in DMEM medium containing 10% fetal bovine serum. Incubator conditions are 37 °C, 5% CO 2 . When the cells are in the logarithmic growth phase, wash the cells three times with PBS, add 1.5 mL of trypsin, digest at 37°C for 4-6 min according to the cell density, discard the trypsin, add DMEM cell growth medium to the culture flask and use The pipette blows the cells into single cell form. A portion of the obtained cell suspension was then evenly distributed into six-well plates with coverslips.

[0042] (2) Liposome transfection

[0043] Transfection was performed when the cells in the six-well plate reached 40-60% confluency. First, mix 4 ug of plasmid DNA with 250 μL of OPTI medium, then mix 10 uL of liposomes with 250 μL of OPTI medium, mix well and let stand for 5 minutes. The...

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Abstract

The invention specifically relates to construction and application of over-expression vectors of three different subtypes of porcine PDLIM3 protein, belonging to the field of bioengineering technology. The nucleotide sequences of the gene coding regions of the three different subtypes of the porcine PDLIM3 protein are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 in a sequence table. Three over-expression vectors of the three subtypes of the porcine PDLIM3 protein can be simultaneously constructed by subjecting the complete coding region fragments of the genes of the three subtypes of the porcine PDLIM3 protein to double enzyme restriction and connecting the treated fragments with the pEGFP-N1 vector having undergone double enzyme restriction. The above-mentioned eukaryotic expression vector construction method is simple and practicable; the over-expression vectors can highly-efficiently enhance expression of the PDLIM3 protein, all contain green fluorescent protein and can be used for detecting expression and location of the three different subtypes of the porcine PDLIM3 protein in eukaryotic cells. The over-expression vectors provided by the invention are also applicable to research on roles of the porcine PDLIM3 protein in growth and development of porcine muscle fibers.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a construction method and application of an overexpression vector of three different subtypes of porcine PDLIM3 protein. Background technique [0002] Actinin-associated LIM protein (PDLIM3) belongs to the PDZ / LIM protein family, which directly binds to α-actinin protein at the Z-line of skeletal muscle, and has recently been found to regulate many myogenic regulatory factors myogenin and MyoD . Therefore, it can be used as a structural protein of muscle fibers and can also promote the development of muscle fibers through transcriptional regulation. Knockout of PDLIM3 can lead to imperfect development of the heart and bring about many muscle-related diseases, so it plays an important role in the development of skeletal muscle and cardiac muscle. Previous studies in humans, mice, chickens and fish reported that there are two distinct transcripts of the PDLI...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85
Inventor 王林杰王薏琳王艳
Owner SICHUAN AGRI UNIV
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