Construction method and application of a cell line stably and highly expressing os-9
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A technology with high stability and high cell lines, which is applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., to achieve stable biochemical properties and facilitate acquisition
Active Publication Date: 2018-01-02
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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[0006] However, there have been no relevant reports on the protective effect of the intestinal mucosal barrier through the overexpression of os-9. Therefore, it is an urgent need to provide a method for the construction and application of a cell line with a stable and high expression of OS-9. question
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Embodiment 1
[0055] S11: Extract the mRNA of the subcultured Caco-2 cells, and perform reverse transcription to obtain cDNA;
[0056] S12: Using the cDNA as a template, perform PCR amplification, electrophoresis and gel cutting recovery to obtain the full-length OS-9 gene;
[0057] In the process of PCR amplification, the primers used include upstream amplification primers, downstream amplification primers and intermediate deletion splicing primers;
[0058] Wherein, the sequence of the upstream primer is shown in SEQ ID NO: 1-SEQ ID NO: 2; the downstream amplification primer is described in SEQ ID NO: 3-SEQ ID NO: 4; the sequence of the middle deletion splicing primer The sequence is shown as SEQ ID NO:5-SEQ ID NO:12.
[0059] In addition, during the amplification process, using the cDNA as a template, the upstream primer, the downstream primer and the intermediate deletion splicing primer are respectively used for amplification to obtain 3 amplification products;
[0060] The three amp...
Embodiment 2
[0072] S21: extract the mRNA of the subcultured Caco-2 cells, and perform reverse transcription to obtain cDNA;
[0073] In this step, the subculture of Caco-2 cells specifically includes:
[0074] Human colon cancer epithelial Caco-2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences; Caco-2 cell complete medium preparation: 20% fetal bovine serum, 1% non-essential amino acid, 1% double antibody, MEM medium as mother solution; Caco- 2 Cell culture conditions: 37°C, 5% CO 2 , saturated humidity, cultured for 4-6 days; when the cells are about 80% confluent, wash the cells 3 times with sterile PBS, add trypsin containing 0.25% EDTA to digest, observe under the microscope, when the cells become round and partly fall off immediately add the medium containing serum to stop the digestion; blow the cell suspension repeatedly with a dropper several times, transfer the cell suspension to a sterile centrifuge tube, put it into a centrifuge, and centrifuge at...
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Abstract
The invention provides a method for constructing a cell line stably and highly expressing OS-9, and belongs to the field of molecular biology technology. The method comprises connecting the plenti6.3 vector and the OS-9 gene, and transforming competent Escherichia coli; performing plasmid extraction on the transformed Escherichia coli to obtain the plenti6.3-OS-9 plasmid; using the plenti6.3-OS The 293T cells were transfected with the ‑9 plasmid, the virus fluid was harvested, and the positive clones were screened after infecting the Caco‑2 cells with the virus fluid. After culturing for 10‑15 days, a cell line with a stable and high expression of OS‑9 was obtained. This cell line facilitates the study of the expression of other functional proteins in the state of OS‑9 overexpression, and provides materials for the study of the relationship between OS‑9 overexpression and the expression of other functional proteins. Quantification and qualification of related proteins can be performed by western blot, immunofluorescence and other operations.
Description
technical field [0001] The invention relates to the technical field of molecular biology, in particular to a construction method and application of a cell line stably and highly expressing OS-9. Background technique [0002] The intestinal mucosal barrier is an important barrier for the body's defense, which can prevent the invasion of pathogenic antigens (bacteria, toxic substances, food antigens, carcinogens, etc.) in the intestinal cavity, keep the internal environment of the body relatively stable, and maintain the normal life activities of the body. [0003] Tight junction proteins between intestinal epithelial cells are an important part of the intestinal mucosal barrier. Once the tight junction protein is destroyed, the permeability between intestinal epithelial cells will increase, and bacteria, endotoxins and macromolecular substances can enter the systemic circulation through the tight junction protein and participate in the occurrence and development of various di...
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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor孙力华杨桦肖卫东王文生
OwnerTHE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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