| The producing of recombinant protein drugs using mammalian cells as host hasbecome the main biological pharmaceutical technology. Among the the currentrecombinant protein in the word, more than80%of them are produced by mammalianhost cells. Compared to the bacteria and yeast, mammalian cells have a poor toleranceability to various environmental stress, and more strict cultivation environment is needto culture it. The large-scale culture of mammalian cells is limited by the above factorsand the cost of producing is higher. On the basis of the mechanism of proliferation,apoptosis, metabolism, etc are indicated. Genetic modifying mammalian cells afterfinding the right target to make it more suitable for cultivation in industry, has been oneof the important research in biological engineering. Combined with our previousinvestigation and the literature, we speculate that serves as molecular stress response-MGF, one of a cytokines producing by cell in stress, is expected to be the target of thehost cell to modify the mammalian host cell in order to increase the ability of stresstolerance. In this study we evaluate the tolerance ability of rCHO-MGF cells to a varietyof stress conditions such as serum-free medium and spent medium after successfullyreconstructing the CHO-MGF host cell. Now introduce the main research contents andresults briefly:1. the sequence of MGF is amplified by PCR, the pLenti6.3_MCS plasmid andMGF is cut by BamHI and acsi enzyme, and the recombinant lenti virus plasmid pL-MGF and the sequence of MGF is connected by T4DNA ligase. At the basis of thesequence is identified, it was packaging using293FT cells, and LV-null and LV-MGFwas obtained after high speed gradient centrifugation. The concentration of LV-null andLV-MGF is1.25*108and2*108.2. More than50%of CHO-K1is obtained after infecting by LV-null and LV-MGFin medium with8μg/ml Polybrene and screened in select medium with8μg/mlblasticidin for two weeks. The expression of MGF in cells is identified by Western blotexperiments, we name the cell expressing MGF as CHO-MGF, and the cell with emptyvirus vector is named CHO-null;3. Serum-free medium, spent medium, and acid/alkaline medium is used asmedium to culture the CHO-K1, CHO-null, CHO-MGF. The stress tolerance of thethree cell are evaluated from the prospect of proliferation, cell cycle, apoptosis and cell vitality and metabolism of glucose and lactic acid:(1) the CHO-MGF in serum-free medium is firstly evaluated. The ability ofCHO-MGF to divide and proliferate is more higher than that of CHO-K1and CHO-nullcell which is determined by MTT experiment and the result of the cell cycle; Accordingto the result of trypan blue dyeing experiments and flow cytometry, the number ofviable CHO-MGF cell comparing with control is more obviously after being culturedfor48h in serum-free medium; After measuring the concentration of glucose and lacticacid using SBA bio-sensor analyzer, the adaptive ability of CHO-MGF to the mediumwith the accumulation of lactic acid is more strong, and the proliferative rate ofCHO-MGF is more quickly because the concentration of glucose is lower and theconcentration of lactic acid is higher in medium culturing CHO-MGF than the control;(2) the CHO-MGF in depleted medium is then evaluated. The medium is made byculturing the CHO-K1cells in spent DMEM/F12medium which is used to simulate thecondition in the later phase of feed-batch culture. The ability of CHO-MGF to divideand proliferate in depleted medium is more higher than that of CHO-K1and CHO-nullcell which is determined by MTT experiment and the result of the cell cycle; Accordingto the result of trypan blue dyeing experiments and flow cytometry, the number ofviable CHO-MGF cell is more than that of CHO-K1and CHO-null after being culturedfor48h in depleted medium; After measuring the concentration of glucose and lacticacid using SBA bio-sensor analyzer, the adaptive ability of CHO-MGF to the mediumwith the accumulation of lactic acid is more strong, and the proliferative rate ofCHO-MGF is more quickly because the concentration of glucose is lower and theconcentration of lactic acid is higher in medium culturing CHO-MGF than the control;(3) The pH of acid/alkaline medium is determined by the result of MTTexperiments. The ability of CHO-MGF to divide and proliferate in acid/alkalinemedium is more higher than that of CHO-K1and CHO-null cell which is determined byMTT experiment and the result of the cell cycle, and this result can be proved by theconcentration of glucose in medium culturing the CHO-MGF is lower than that of thecontrol; According to the result of trypan blue dyeing experiments and flow cytometry,the over-expression of MGF can improve the ability of anti-apoptosis of mammalianhost cell in acid/alkaline medium which can also be proved by the result of trypan bluefrom dyeing experiments.In summary, we construct a stable expression of MGF CHO K1celllines-CHO-MGF using lenti virus. Compared with the control, the CHO-MGF cell is more adaptive to the serum-free culture, spent medium, and weak acid medium. So thatto say, MGF, have a certain anti-apoptosis ability. |
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