中文摘要：

目的构建携带大鼠白细胞介素-6（interleukin 6，IL-6）基因的重组腺病毒，为研究IL-6在神经损伤中的生物学作用提供技术手段。方法体外扩增大鼠IL-6基因，定向克隆到pAdTrace—TOX腺病毒穿梭质粒中，构建重组pAdTrace—IL-6过表达腺病毒质粒，并与骨架质粒pad—Easy一1在BJ5183菌中发生同源重组．获得pAd—IL-6腺病毒载体，经PacI酶切后转染HEK293细胞进行包装扩增。通过Ad—IL-6腺病毒感染PCI2细胞，Real—timePCR和Westernblot检测PCI2细胞中IL一6及STAT3的表达。结果PCR电泳及酶切、测序鉴定均证实目的基因IL-6正确克隆至腺病毒载体中，所构建的腺病毒Ad—IL-6可感染PCI2细胞，有效增加IL-6的表达水平，促进IL_6信号通路中关键蛋白磷酸化STAT3的表达。结论成功构建携带IL-6的重组腺病毒载体，该载体可显著增高PCI2细胞中IL-6基因和蛋白表达水平，并上调IL-6相关信号通路。

英文摘要：

We aimed to construct recombinant adenovirus containing specific rat interelukin-6 （IL-6） gene, providing a technological method for the study of IL-6 functions during nerve injury. Rat IL-6 gene was amplified by PCR and then cloned into the pAdTrace-TOX adenovirus shuttle vector to construct recombinant pAdTrace-IL- 6 adenovirus plasmid. The pAdTrace-IL-6 was homologously recombined with backbone vector pAd-Easy-1 in E. coli BJ5183 to form the adenovirus vector pAd-IL-6. After linearized by Pac I, the pAd-IL-6 was transfected into HEK 293 cells to package and amplify the recombinant adenovirus Ad-IL-6. PC12 cells were infected with the Ad-IL-6, and the mRNA and protein expression levels of IL-6 and STAT3 in PC12 cells were detected by Real- time PCR and Western blotting, respectively. The IL-6 gene specific for rat was confirmed to be correctly cloned into an adenovirus vector via the methods of PCR electrophoresis, enzyme digestion and sequence analysis. The Ad-IL-6 was able to effectively infect PC12 ceils and significantly upregulate the mRNA and protein expression level of IL-6. Meanwhile, the level of p-STAT3 expression, a down stream target protein in IL-6 signal transduction pathway, was increased in the Ad-IL-6 infected PC12 cells. Therefore, our data suggested that recombinant adenovirus vector pAd-IL-6 was successfully constructed, which could effectively increase the expression level of IL-6 and activate IL-6 pathway through upregulating p-STAT3 expression.