摘要
目的构建实验所需的真核表达载体，特别是人源性tRNA val启动子驱动CTE介导的核酶高效表达载体pPHCV5-CR以及对应的无CTE介导的载体pPHCV5-R；建立HCV Subgenomic Replicon稳定转染和表达的细胞株；探讨核酶和核酶-CTE复合物对丙型肝炎病毒亚基因组的影响，进一步探讨丙型肝炎治疗研究方向。
研究方法
1、构建相应的核酶载体
合成CTE-DNA，设计、构建含tRNAval启动子的核酶和带有CTE的含tRNAval启动子的核酶。
2、建立HCV Subgenomic Replicon稳定转染和表达的细胞株
在脂质体lipofectamine TM2000介导下，将含有HCV Subgenes的真核表达质粒pHCV BM4-5导入人肝癌细胞系QSR7701中，经G418筛选抗性克隆，扩大培养，建立转染克隆细胞系。用反转录聚合酶链反应(RT-PCR)和蛋白印迹法证实转染成功及其蛋白表达。
3、观察四种质粒pPHCV5-R1、pPHCV5-R2、pPHCV5-CR1、pPHCV5-CR2对HCV Subgenomic Replicon稳定转染和表达的细胞株中HCV RNA和相应病毒蛋白表达的影响。
结果
1、经测序后证实，成功构建相应的核酶载体，为下一步实验奠定基础。
2、经RT-PCR和Western Blot证实，成功建立稳定转染HCV
Subgenomic Replicon和表达的细胞株。
3、用构建的普通核酶pPHCV5-R1、pPHCV5-R2和复合核酶pPHCV5-CR1、pPHCV5-CR2瞬时转染含HCV亚基因复制子的稳定转染QSG7701细胞株，48小时后收集细胞进行RT-PCR和WesternBlot，RT-PCR(以β-actin为内对照)结果显示：pPHCV5-CR1转染组未见明显扩增目的条带，pPHCV5-R1转染组可见扩增目的条带，亮度低于未转染组和空质粒转染组；pPHCV5-CR2转染组可见扩增目的条带，亮度低于未转染组和空质粒转染组，pPHCV5-R2转染组可见扩增目的条带，亮度与未转染组和空质粒转染组接近。这些提示：复合核酶(带有CTE)在细胞内的切割靶RNA的效率明显高于普通核酶。普通核酶难以切割的位点，复合核酶能进行有效的切割；普通核酶能进行切割的位点，带CTE-核酶的切割效率明显提高。WesternBlot(以β-actin为内对照)结果显示：应用软件分析各组细胞目的蛋白表达量无明显差异，可能与瞬时转染作用时间短、蛋白表达变化不明显，也可能与实验重复次数少、可用数据少或者检测方法有关。
结论新型核酶(带有CTE)在细胞内的切割靶RNA的效率明显高于普通核酶。普通核酶难以切割的位点，带CTE-核酶能进行有效的切割；普通核酶能进行切割的位点，带CTE-核酶的切割效率明显提高。
Objective To construct ribozyme high-performance expression eukaryotic vector with engineered tRNAval promotor mediated by CTE(constitutive transport element) or not; To construct a cell line transfected and expressed stably with HCV Subgenomic Replicon; To study the inhibition effect of ribozyme / ribozyme-CTE on intracellular HCV mRNA, which may establish a new therapeutic approach for hepatitis C.
Methods
1、To construct ribozyme expression eukaryotic vectorFirst, to synthesize CTE-DNA; Then, to design and construct the ribozymes with tRNAval promoter as well as the ribozymes with both tRNAval promoter and CTE.
2、To construct a cell line transfected and expressed stably with HCV Subgenomic Replicon
Using Lipofectmine~(TM)2000, the plasmid P~(HCV BM4-5) with HCV Subgenomic Replicon and p~(RC/CMV) were transfected into QSG7701 cells respectively. Then the cell line with stable expression of HCV Subgenomic Replicon was obtained after the G418 screening, and non-transfected QSG7701 cells as control. It was verificated by RT-PCR and Western Blotting.
3、To study the effect of pPHCV5-R1,pPHCV5-R2,pPHCV5-CR1 as well as pPHCV5-CR2 on HCV Subgenomic Replicon and the corresponding protein.
Result
1、Construct ribozyme expression eukaryotic vector successfully, which was verificated by sequencing.
2、Construct cell lines transfected and expressed stably with HCV Subgenomic Replicon, which was verificated by RT-PCR and Western Blotting.
3、The plasmids that contain these classic ribozyme genes and new ribozyme genes were transfected into QSG7701 cells using Lipofectmine~(TM)2000．The following experiments were carried out after 48 hours of transfection. RT-PCR and WesternBlotting were used to detect HCV RNA and corresponding protein respectively. The results of RT-PCR suggested that those new rebozymes with CTE could cut target RNAs more effective than classic ribozymes without CTE. Those new ribozymes with CTE could cut target RNAs that classic ribozymes could not cut. The results of WesternBlotting suggested that the quantity of HCV protein didn't change markedly, which may be due to low detection efficiency or limited data available.
Conclusion New combined ribozymes with CTE was more effective than rebozymes without CTE.
引文
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