目的 构建肌浆网-内质网钙离子转运ATP酶(SERCA)基因的干扰慢病毒表达载体,建立稳定转染的大鼠肾上腺嗜铬细胞瘤(PC 12)细胞系。方法 人工设计、合成针对SERCA基因干扰序列,退火后连接到PGLV3干扰载体上,与pGag/Pol、pRev、pVSV-G共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度,感染PC 12细胞,建立稳定细胞株;应用RT-PCR和Western blot技术检测PC 12稳定细胞中SERCA基因和蛋白表达情况,并与对照组进行比较。结果 成功构建了针对SERCA基因的RNAi慢病毒表达载体,病毒滴度为3×108 U·mL-1;建立稳定转染的PC 12细胞株。有效干扰验证显示,shSERCA能明显降低SERCA的mRNA 及蛋白水平(P<0.01)。结论 成功构建SERCA基因的shSERCA慢病毒表达载体,建立稳定干扰SERCA表达的PC 12细胞株。
Abstract
OBJECTIVE To construct the SERCA gene interference lentivirus expression vector and establish stable transfected PC 12 cell line. METHODS The interference sequence targeting at rat SERCA gene was designed and synthesized. pGag/Pol, pRev, and pVSV-G were co-transfected into 293T cells. The lentivirus particles were packaged and generated. The virus titer was detected. PC 12 cells were transfected for establishing the stable cell line; RT-PCR and Western blot were used to detect SERCA gene and protein expression in stable PC 12 cells,and the RESULTS were compared with those in the control group. RESULTS The lentivirus expression vector targeted at SERCA was successfully constructed and the virus titer was 3×108 U·mL-1. Stable transfected PC 12 cells line was established. The effective interference verification revealed that shSERCA could significantly reduce the mRNA and protein levels of SERCA (P<0.01). CONCLUSION The shRNA lentiviral expression vector of SERCA gene is successfully constructed and the PC 12 cell line stably interfering with SERCA expresion is established.
关键词
肌浆网-内质网钙离子转运三磷酸腺苷酶基因 /
核糖核酸干扰 /
大鼠肾上腺嗜铬细胞 /
钙离子转运ATP酶 /
慢病毒
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Key words
SERCA gene /
RNA interference /
PC 12 cell /
sarco-endoplasmic reticulum Ca2+-transporting ATPase /
lentivirus
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金项目资助(81573643)
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