摘要
目的采用荧光定量PCR法检测转染后可分泌表达人凝血因子ⅦCHO细胞株CHO-FⅦ中FⅦ基因的拷贝数。方法提取CHO-FⅦ和CHO-pcDNA3.1(空载体转染得到的细胞株)基因组DNA,分别以含有β-actin基因片段的质粒pMDT-actin和含有FⅦ全长cDNA序列的质粒pcDNA-FⅦ制作标准曲线,以CHO-FⅦ和CHO-pcDNA3.1的DNA为模板进行qPCR反应,计算出模板中细胞总数以及目的基因的拷贝数,得到每个细胞中目的基因的拷贝数,反应均在ABI Stepone Plus荧光定量PCR仪上进行。结果经检测模板中的细胞数为24 421±7 414,FⅦ基因的拷贝数49 455±3 502,每个细胞含有2个FⅦ基因拷贝,空载体转染得到的细胞株中并没有检测到FⅦ基因的存在。结论 FⅦ基因是以2个拷贝的比例整合到宿主染色体上的。
Objective To detect the copy numbers of Human Coagulation Factor Ⅶ（ FⅦ） gene in stable expression CHO cell line with fluorescent quantitative polymerase chain reaction（ qPCR）. Methods The quantitive genomic DNA of plasmid pMDT-actin and pcDNA-FⅦ were used as templates for making standard curves,respectively. The result was analyzed by Stepone software V2. 2 to calculate the total cell number and the copy number of FⅦ gene in templates. All of the qPCR reactions were performed in ABI Stepone plus system. Results The total cell number in templates is 24 421 ± 7 414.The copy number of the FⅦ gene is 49 455 ± 3 502. No specific gene amplication can be detected in the CHO-pcDNA3. 1.Conclusion The result clearly showed that every CHO cell harbored two FⅦ gene copies.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2014年第6期602-605,共4页
Chinese Journal of Blood Transfusion
关键词
人凝血因子Ⅶ
绝对定量
实时荧光定量PCR
拷贝数
human coagulation factor Ⅶ
absolutequantification
real-time fluorescent quantitative PCR
copy nmn-ber
作者简介
林方昭（1966．12-），女，副主任技师，主要从事血栓与止血相关研究，电话：028-61648541，通信作者：E-mail：fangzhao@126．com
