shRNA沉默CBS基因的慢病毒载体构建及稳定转染PC12细胞系的建立

摘要

目的：硫化氢是一种重要的气体信号分子，作为一种神经调质在神经系统中起重要作用。胱硫醚-β-合成酶（CBS）是脑内硫化氢合成的主要酶。构建针对大鼠 CBS 基因的 shRNA 干扰载体，稳定转染 PC12细胞，观察该载体对 PC12细胞 CBS 基因的沉默效应。方法：构建三条针对大鼠 CBS 基因的 shRNA，经前期实验筛序一条最有效靶点与载体 GV248（hU6-MCS-Ubiquitin-EGFP-IRES-puromycin）连接，经转化及 PCR 阳性克隆筛选及测序鉴定。将 LV-CBS-ShRNA 慢病毒载体连同包装载体经脂质体2000共转染到293T 细胞，慢病毒包装后用荧光法进行滴度测定。将包装好的慢病毒转染到 PC12细胞，用嘌呤霉素进行筛选，得到稳定转染 LV-CBS ShRNA的 PC12细胞。实时荧光定量 PCR 检测 CBSmRNA 的表达，Western-blot 检测 CBS 蛋白的表达。结果：PCR 扩增和测序结果证明，成功构建大鼠 LV-CBS ShRNA 慢病毒载体，经包装产生的慢病毒滴度为1×109 TU ／mL。与转染阴性对照慢病毒（LV-NC-ShRNA）的细胞比较，LV-CBS ShRNA 慢病毒转染可使 PC12的 CBSmRNA 和 CBS 蛋白表达分别下降51．2％和48％。成功构建 CBS 基因 ShRNA 干扰的 PC12细胞株，为后续研究 CBS 在神经系统中的作用奠定基础。%Objective:Hydrogen sulfide gas is an important signaling molecule.As a neuromodulator,it plays an impor-tant role in the nervous system.Cystathionine-beta-synthase (CBS)is the main synthesis enzyme of hydrogen sulfide in the brain.To construct the lentivirus with shRNA interference vector for CBS gene by RNA interference technology,lenti-virus was packaged,then transduced into PC12 cells.Method:Three shRNA sequences targeting CBS gene were de-signed and synthesized,and the best one was cloned into GV248 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin)vector to construct the lentiviral expression vector containing CBS ShRNA (LV-CBS-ShRNA).Positive clones confirmed by PCR and DNA sequencing.Subsequently LV-CBS-ShRNA and packing plasmids were contransfected into 293T cells by Lipofectamin 2000.The titer of virus was detected according to the expression of enhanced green fluoreseen protein (EGEP).The packaged lentivirus transfected into PC12 cells were filtered with puromycin,then the PC12 cells with CBS ShRNA transfected were constructed.Real-time PCR detected the expression of CBS mRNA.Western-blot detected the expression of protein CBS.Results:PCR analysis and DNA sequencing proved that LV-CBS-ShRNA was constructed successfuly.The lentivirus titer is,1 ×109 TU /mL.Compared with negative control lentivirus(LV-NC ShRNA)the level of CBS mRNA and CBS protein in LV-CBS-ShRNA decreased 51.2% and 48% respectively.Conclusion:PC12 cells with CBS ShRNA strain was successfully constructed by using lentiviral vector,and provided a stable cell lines to study the effect of H2 S on nervous system.