A cell line overexpressing ciapin1 protein and its preparation method and application

A technology of BHK-21CIAPIN1 and cell lines, applied in the field of biotechnology and cytogenetics, to achieve the effects of easy storage, increased incidence and stable properties

Active Publication Date: 2018-01-23
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] At present, the reports on CIAPIN1 research are still very limited, mainly focusing on tumor-related aspects. However, these results have proved that CIAPIN 1 has an important regulatory effect on cell proliferation and differentiation, and plays an important role in cell apoptosis and anti-apoptosis

Method used

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  • A cell line overexpressing ciapin1 protein and its preparation method and application
  • A cell line overexpressing ciapin1 protein and its preparation method and application

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preparation example Construction

[0028] A preparation method for overexpressing CIAPIN1 protein cell line, comprising the following steps:

[0029] 1) Construction of recombinant eukaryotic expression plasmid: RT-PCR amplified the cDNA sequence of CIAPIN1 protein and inserted it into the eukaryotic expression plasmid pIRESneo to obtain the recombinant eukaryotic expression plasmid pIRES-CI;

[0030] 2) Transfection: Transfect the above-mentioned recombinant eukaryotic expression plasmid into BHK-21 cells;

[0031] 3) Screening: the transfected cells were screened with G418-containing culture medium;

[0032] 4) Obtain an immortalized cell line overexpressing CIAPIN1 protein: the screened and cultured cells were subjected to limiting dilution to obtain positive monoclonal cells, among which the cell line capable of overexpressing CIAPIN1 protein was the immortalized cell line overexpressing CIAPIN1 protein.

[0033] Preferably, the cDNA sequence encoding CIAPIN1 protein in step 1) is shown in SEQ ID NO:1.

...

Embodiment 1

[0041] Example 1 Construction and sequencing of recombinant pMD-CI cloning vector

[0042] 1. Design and synthesis of oligonucleotide primers

[0043] According to the nucleotide sequence of CIAPIN1 in GenBank, primers for cloning CIAPIN1 cDNA were designed and synthesized:

[0044] CIAPIN1-F: 5’- GATATC ATGGAGGAGTTTGGGATCTCC-3' (the underlined part is the EcoR V restriction site);

[0045] CIAPIN1-R: 5’- GAATTC CTAGGCATCCTGGAGATTGCTAT-3' (the underlined part is the EcoR I restriction site).

[0046] 2. Extraction of total RNA from BHK-21 cells

[0047] Add 1mL Trizol directly to the BHK-21 cells (baby hamster kidney cells, Baby Hamster Syrian Kindey) in a 50ml cell culture flask, place at room temperature for 5min to fully lyse, centrifuge at 12 000r / min for 5min, take the supernatant, and use 200μL chloroform / mL Add chloroform to Trizol, oscillate and mix well, place at room temperature for 15 min, centrifuge at 12 000 r / min at 4°C for 15 min, aspirate the supernatant...

Embodiment 2

[0052] Example 2 Construction of recombinant eukaryotic expression vector pIRES-CI

[0053] 1. Recycling of target fragments

[0054] Weigh 1g of agarose and dissolve in 100mL 1×TAE by heating; add EB with a final concentration (V / V) of 0.1% when cooled to 40-50°C, mix well and pour the gel; insert the gel back into the tooth comb, cool at room temperature for later use ; Add the loading mixture (90 μL CIAPIN1 cDNA PCR product + 10 μL 10×Loading Buffer) to the loading well. After electrophoresis at a voltage of 5V / cm for the required time, take out the gel, observe it on a transmission ultraviolet lamp, or take a picture. Cut out the target band under ultraviolet light, freeze and thaw three times, crush, extract with phenol and chloroform respectively, precipitate with absolute ethanol, wash with 75% ethanol, dissolve in an appropriate amount of water, and store at -20°C for later use.

[0055] 2. Ligation reaction between target fragment and eukaryotic expression vector pI...

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Abstract

The invention discloses a cell line overexpressing CIAPIN1 protein and its preparation method and application; the cell line name is BHK-21 CIAPIN1+, the preservation number is CCTCC C2014149, and the amount of the expressed CIAPIN1 protein is 10 times that of normal BHK-21 cells. times; the preparation method of this cell line is to insert the cDNA sequence of CIAPIN1 into the eukaryotic expression plasmid pIRESneo, and then transfect BHK‑21 cells; after being screened by G418-containing culture medium, the cells are obtained by limiting dilution method to obtain positive monoclonal cells, namely Cell lines overexpressing CIAPIN1 protein were immortalized. The cultivation of the rabies virus by the cell line of the present invention can significantly affect the titer of the virus, and is of great significance to the efficient cultivation of the virus and the production of the inactivated vaccine. The cell line of the present invention has a good application prospect in the cultivation of the rabies virus.

Description

technical field [0001] The invention belongs to the fields of biotechnology and cytogenetics, and relates to a cell line overexpressing CIAPIN1 protein, a preparation method and application thereof. Background technique [0002] CIAPIN 1 (cytokine induced apoptosis inhibitor 1), also known as anamorsin, is a newly discovered molecule related to apoptosis. CIAPIN 1 is located on the long arm of chromosome 16 in the human gene. It consists of 8 exons and 7 introns, with a relative molecular mass of 39,000. A genericmethyltransferase profile domain was found at the 60-99 amino acid position of the N-terminus, the partial sequence of the C-terminus was homologous to the conserved gene COG5636, and the Cx2Cx24Cx2C zinc band domain was found at 246-277 amino acids. CIAPIN 1 is located on chromosome 8 in the mouse gene, the mature mRNA length is 1515bp, and the CDS length is 929bp. [0003] Shibayama et al. reported for the first time in 2004 that this molecule is an anti-apopto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12N7/00
Inventor 王晓虎向华黄元陈晶陈远媚
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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