pCDNA3.1-Annexin A7载体的构建及在Hca-p细胞中的表达

Construction of pCDNA3.1-Annexin A7 and expression in Hca-p

关键词:  膜联蛋白A7  PCR  转染  肿瘤  淋巴道转移

Key words:  annexin A7  PCR  transfection  hepatocarciroma

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cDNA，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400 μg/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-blotting 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-Annexin 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。

Abstract:

［Objective］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.