摘要
目的建立胱硫醚-β-合成酶(CBS)基因过表达PC12细胞株并进行鉴定。方法根据Gen Bank数据库中的CBS c DNA序列设计合成CBS基因引物,PCR扩增CBS基因,并将其克隆到慢病毒过表达载体GV341中,用慢病毒过表达载体连同两种包装病毒载体对293T细胞进行转染,收获含CBS慢病毒载体的上清液,用其感染正常的PC12细胞,用嘌呤霉素进行筛选得到CBS基因过表达PC12细胞株。用实时荧光定量PCR法检测所得PC12细胞(观察组)、空载体转染的PC12细胞(对照组)、正常PC12细胞(空白组)中的CBS mRNA。用Western blot法检测上述三组细胞中的CBS蛋白。结果观察组、对照组、空白组PC12细胞CBS mRNA表达量分别为162.20±11.20、1.00±0.03、2.30±0.07,CBS蛋白表达量分别为1.53±0.09、1.00±0.10、0.96±0.10。观察组PC12细胞CBS mRNA、蛋白表达量均高于对照组和空白组(P均<0.05),对照组PC12细胞CBS mRNA、蛋白表达量与空白组相比差异均无统计学意义。结论成功构建CBS基因过表达PC12细胞株。
Objective To establish stable PC12 cell line with overexpression of cystathionine-β-synthase （CBS）and to identify it.Methods CBS primers were designed according to cDNA sequence of CBS gene from GenBank.The CBS gene was amplified using PCR and ligated into the lentiviral vector GV341.293T cells were transfected with the recombi-nant vector and two kinds of packaging viral vector.Viral supernatant was collected and was used to transfect PC12 cells. After puromycin screening,PC12 cells with CBS gene transfection were constructed.The CBS mRNA and protein of the PC12 cells and the empty vector transfected PC12 cells was detected by real-time fluorescent quantitative PCR and Western blotting,respectively.Results The CBS mRNA expression in the observation group,control group and empty vector group was 162.2 ±11.2,1.0 ±0.03 and 2.3 ±0.07,respectively.The CBS protein expression in the observation group,control group and empty vector group was 1.53 ±0.09,1.00 ±0.10 and 0.96 ±0.10,respectively.The CBS mRNA and protein expression levels of the observation group were higher than those of the control group and empty vector group （P 〈0.05）. The difference of CBS mRNA and protein expression between the control group and empty vector group was not statistically significant.Conclusion The PC12 cell line with overexpressed CBS is successfully constructed.
出处
《山东医药》
CAS
北大核心
2016年第26期17-19,共3页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81200986)
国家级大学生创新创业训练计划项目(201310555196)
