【摘要】 [目的]构建pCDNA3.1-Annexin A7载体并稳定转染Hca-p细胞。[方法]提取小鼠总RNA,逆转录成cD-NA,PCR扩增,获取Annexin A7 CDS,将PcDNA3.1(+)质粒和Annexin A7 CDS片段行BamH I和EcoR I双酶切,构建并抽提pCDNA3.1-Annexin A7质粒,测序鉴定后转染Hca-p细胞,经400μg/mL G418完全培养基筛选获得pCDNA3.1-Annexin A7载体稳定转染的Hca-p细胞,在蛋白质水平检验Annexin A7的表达并行基因组DNA鉴定。[结果]成功构建pCDNA3.1-Annexin A7载体,经测序鉴定,所得到的载体中的CDS经比对与库中序列一致。pCDNA3.1-Annexin A7载体转染Hca-p细胞后,获得稳定上调表达Annexin A7的Hca-p细胞株;行基因组DNA鉴定,证实pCDNA3.1-Annexin A7已转入Hca-P细胞中,western-b lotting结果显示转染前后蛋白的表达相对值Annexin A7/GAPDH分别为0.43544±0.0112和0.45957±0.0065(P<0.05)。[结论]pCDNA3.1-An-nexin A7的构建并稳定转染Hca-p细胞成功,为通过上调Annexin A7在Hca-P的表达以证实Annexin A7与恶性肿瘤淋巴道转移潜能关系的相关研究打下了基础。更多还原

【Abstract】 [Objiective] To construct pCDNA3.1-Annexin A7 and to transfectant it the Hca-P stably.[Methods]The Annexin A7 gene was amplified by PCR.BamH I and EcoR I enzyme were used to digest the Annexin A7 gene and PcDNA3.1 plasmid.The pCDNA3.1-Annexin A7 were constructed to transfectant to the Hca-P stably.The effection of transfectanting was detected by genome DNA checkup and Western-blotting.[Results] The DNA sequencing result showed that the pCDNA3.1-Annexin A7 was constructed successfully,Genome DNA checkup demonstrated that the Hca-P were transfectanted by the pCDNA3.1-Annexin A7 stably,Western-blotting indicated that the value of optical density Annexin A7/GAPDH in transfectanted Hca-P was significantly higher than that in normal Hca-P(0.43544 0.0112 vs 0.45957±0.0065,P<0.05).[Conclusions] pCDNA3.1-annexin A7 was constructed and transfectanted to the Hca-P successfully.更多还原