大鼠白细胞介素-6重组腺病毒构建及初步鉴定

Construction and preliminary identification of rat interleukin-6 recombinant adenovirus

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-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PC12细胞,Real-time PCR和Western blot检测PC12细胞中IL-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PC12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PC12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。

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.