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目的:获取人组氨酸磷酸酶蛋白PHP14基因,并构建其N端和C端GFP融合的真核表达载体,建立过表达PHP14的NIH-3T3细胞系,并观察其对NIH-3T3细胞体外增殖和非锚定依赖性生长的影响。方法:以HeLa cDNA为模板,PCR扩增PHP14的全长编码基因,分别克隆到pEGFP-N2和pEGFP-C3载体中,构建pEGFP-N2-PHP14和pEGFP-C3-PHP14真核表达载体,利用脂质体将构建的载体转染到NIH-3T3细胞中,MTT法检测细胞增殖,软琼脂成集落法检测体外非锚定依赖性生长能力。结果:成功构建了过表达PHP14的真核表达载体pEGFP-N2-PHP14和pEGFP-C3-PHP14,并在NIH-3T3细胞中检测到了目的基因的过表达,PHP14在NIH-3T3细胞中过表达并不影响NIH-3T3细胞的体外增殖,但赋予了NIH-3T3细胞非锚定依赖性生长的能力。结论:成功构建了过表达PHP14的NIH-3T3细胞模型,在NIH-3T3细胞中过表达PHP14并不影响NIH-3T3细胞的体外增殖,但赋予了NIH-3T3细胞非锚定依赖性生长能力。
OBJECTIVE: To obtain human recombinant human phosphatase PHP14 gene and construct an eukaryotic expression vector containing N-terminal and C-terminal GFP fusion. NIH-3T3 cell line overexpressing PHP14 was established and the proliferation of NIH-3T3 cells was observed in vitro And non-anchored dependent growth. Methods: HeLa cDNA was used as a template to amplify the full-length coding gene of PHP14 and cloned into pEGFP-N2 and pEGFP-C3 vector respectively to construct eukaryotic expression vector pEGFP-N2-PHP14 and pEGFP-C3-PHP14. The vector was transfected into NIH-3T3 cells by plastid. Cell proliferation was detected by MTT assay. Non-anchorage-dependent growth in vitro was detected by soft agar assay. Results: The eukaryotic expression vectors pEGFP-N2-PHP14 and pEGFP-C3-PHP14 that overexpressed PHP14 were successfully constructed and the over-expression of the target gene was detected in NIH-3T3 cells. The overexpression of PHP14 in NIH-3T3 cells Does not affect in vitro proliferation of NIH-3T3 cells, but confers a non-anchorage-dependent growth on NIH-3T3 cells. CONCLUSION: NIH-3T3 cell model overexpressing PHP14 was successfully constructed. Overexpression of PHP14 in NIH-3T3 cells did not affect in vitro proliferation of NIH-3T3 cells, but conferred non-anchorage-dependent growth on NIH-3T3 cells.