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AIM: To obtain high level expression of recombinant human truncated osteoprotegerin (TOPG) with higher bioactivity in CHO-DHFR~-cells. METHODS: The recombinant vector pcDNA3.1/DHFR-TOPG was constructed and transfected into CHO-DHFR~- Cells by the directions of LipofectAMINE~(TM)2000 for stable expression. The stable expression cell strains were screened by selective medium IMDM with 50 mL/L FCS, then serially passed in methotraxate (MTX) for gene amplification. The expression were analyzed by ELISA and RT-PCR. At last, the bioactivity analysis was performed in vitro. RESULTS: The expression level of recombinant truncated human OPG was up to 6 mg/L·72 h, and it had significant suppression effect on the formation of OLC(P<0.05). CONCLUSION: Recombinant truncated human OPG has high expression and bioactivity. The results make it possible for further studying and clinical implying of OPG.
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Objective: To express glycosyl-phosphatidylinositol (GPI) modified Met- RANTES fusion protein on Chinese hamster ovary (CHO) cells and to develop a novel immunosuppressant GPI anchored form of Met-RANTES. Methods: The eukaryotic expression vector PEF/GPI-Met-RANTES were constructed and transfected into CHO cells by electroporation. The transfectants were selected with methotrexate (MTX). Expression of the recombinant protein was assessed by flow cytometric analysis, cell immunofluorescence staining and immunogold electron microscopy. Results: The chimeric molecules of GPI anchored form of Met-RANTES including the whole reading frame were constructed, and the sequence was identical to the designed sequence. GPI anchored form of Met-RANTES was stably expressed on CHO- DHFR- cells. Conclusion: A large amount of GPI modified Met-RANTES fusion protein was expressed on CHO cells. GPI anchored form of Met-RANTES may be used as novel immunosuppressant for suppressing reaction in graft rejection.
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@#ObjectiveTo validate the feasibility of the outer space carrying system in culturing the CHO(dhfr-)cells in hypothermia, and to observe the effects of this carrying system on the growth characteristics of the CHO(dhfr-) cells.MethodsThe growth characteristics of the CHO(dhfr-) cells were observed and analyzed with cell morphological observation, MTT assay, FCM,3H incorporation and chromosome after the cells were cultured for 25 days in the carrying system. ResultsComparing with the control group, the CHO(dhfr-) cells appeared multiple cell morphological changes, the difference of cell cycle was not significant, the broken chromosome was not seen, the cell growth speed decreased markedly and big molecular biosynthesis increased obviously. ConclusionThe outer space carrying system has no outstanding effects on the survival and heredity of the CHO(dhfr-) cells, so that it can be used in cell carry.
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@#ObjectiveTo investigate the changes of the morphology, growth and cycle of CHO(dhfr-) cells after space flight.MethodsCHO(dhfr-) cells were carried in the No.18 recoverable satellite and monocloned harvesting cells before multiplying. 4 cell lines were selected randomly,and the growth characteristics of the most slowly growing one at the fifth passage was observed by methods of MTT and FCM as well as the cells' shape.Results159 cell strains were obtained after monocloning and multiplying. The cells' morphology changes, growth speed decrease and the number of G1 phase increased markedly.ConclusionSpace flight induced morphological changes of cells and it is impossible to screen out finer bioengineering cells.