论文部分内容阅读
目的构建特异性针对大鼠TLR4基因的siRNA重组腺病毒载体,为进一步研究TLR4在不同疾病中的免疫调节机制奠定重要基础。方法设计并合成3对大鼠TLR4基因的siRNA序列,退火处理后定向克隆到pSES-HUS穿梭质粒中,获得pSES-HUS-siTLR4,经Pme I线性化后与pAdEasy-1骨架质粒在BJ5183细菌中进行同源重组,从而构建pAd-siTLR4质粒,通过脂质体转染,在HEK293细胞中包装形成Ad-siTLR4腺病毒颗粒,用该病毒感染PC12细胞,从基因和蛋白质水平分别检测3对siTLR4的抑制效果,同时从蛋白质水平检测TLR4下游关键分子NF-κB的表达。结果 PCR、酶切和测序均证实目的基因正确克隆到所构建的pAd-siTLR4重组腺病毒载体中;经包装获得的Ad-siTLR4病毒颗粒在PC12细胞中能够有效地抑制TLR4 mRNA和蛋白水平的表达,并显著地降低了NF-κB的表达。结论成功构建了pAd-siTLR4重组腺病毒质粒,同时包装获得了Ad-siTLR4重组腺病毒,转染PC12细胞后不仅能明显降低TLR4的表达,而且有效地抑制了TLR4通路下游关键分子NF-κB的表达。
Objective To construct siRNA recombinant adenovirus vector targeting rat TLR4 gene and lay a foundation for further study of TLR4 immunomodulatory mechanism in different diseases. Methods The siRNA sequences of 3 pairs of TLR4 genes were designed and synthesized. After annealed, pSES-HUS-siTLR4 was cloned into pSES-HUS shuttle plasmid. After linearized by Pme I, the pAdEasy-1 backbone plasmid was ligated into BJ5183 Then the recombinant plasmid pAd-siTLR4 was constructed and transfected into HEK293 cells to construct Ad-siTLR4 adenovirus vector. The virus was used to infect PC12 cells. Three pairs of siTLR4 Inhibit the effect, and detect the expression of NF-κB, a key molecule downstream of TLR4, at the protein level. Results PCR, digestion and sequencing confirmed that the target gene was correctly cloned into the constructed recombinant adenovirus vector pAd-siTLR4; Ad-siTLR4 virus particles obtained in packaging PC12 cells can effectively inhibit the expression of TLR4 mRNA and protein levels , And significantly reduced the expression of NF-κB. Conclusion The recombinant adenoviral plasmid pAd-siTLR4 was successfully constructed and the Ad-siTLR4 recombinant adenovirus was successfully constructed. The transfection of PC12 cells not only significantly reduced the expression of TLR4, but also inhibited the expression of NF-κB, a key factor downstream of TLR4 pathway expression.