Construction method and applications of cell strain capable of expressing OS-9 stably and highly
A technology of OS-9 and construction method, which is applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., to achieve the effect of facilitating acquisition and stabilizing biochemical properties
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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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