小鼠雌激素受体α基因重组腺病毒载体的构建及鉴定

Construction and analysis of the recombined adenovirus carrying mouse estrogen receptor α gene

目的:构建C57BL/6小鼠雌激素受体α(Esr1)重组腺病毒表达载体,扩增并纯化重组腺病毒颗粒.方法:采用RT-PCR方法从C57BL/6小鼠卵巢细胞扩增目的Esr1 CDS区基因片段,至pMD19-TSimple载体后测序鉴定.亚克隆Esr1CDS区基因片段至腺病毒穿梭质粒pDNR-CMV,再与骨架质粒pLP-Adeno-X-CMV在感受态大肠杆菌ElectroMAXTMDH10BTMCells中进行同源重组获得腺病毒载体质粒Adeno-Esr1,PacI酶切线性化后脂质体法转染HEK293细胞进行包装、扩增,测定感染性滴度,获得重组腺病毒Ad-Esr1.将病毒感染HEK293细胞,Western Blot检测Esr1蛋白表达.结果:测序证实提取的Esr1 CDS区基因序列完全正确;测序及酶切鉴定均表明重组腺病毒载体构建成功;感染性滴度为6.4×1013(pfu/L),Western Blot检测Ad-Esr1感染的HEK293细胞能表达Esr1蛋白,而未感染的HEK293细胞蛋白不表达.结论:成功构建了C57BL/6小鼠Esr1基因重组腺病毒载体,为进一步研究Esr1在神经系统中生物学功能及对神经系统疾病的基因治疗奠定了基础.

AIM：To construct recombinant adenovirus vector carrying C57BL/6 mouse estrogen receptorα（Esr1） gene,and further to propagate and purify the virus particles.METHODS：The CDS fragment of Esr1 gene was amplified by RT-PCR from C57BL/6 mouse ovary cells and put into pMD19-T Simple vector,then verified by sequencing.The fragment was subcloned into the adenovirus shuttle plasmid pDNR-CMV.The recombinated shuttle plasmid was homogenously recombined with pLP-Adeno-X-CMV in ElectroMAXTM DH10BTM Cells and the recombinant adenoviral plasmid Adeno-Esr1 was generated.Then plasmid Adeno-Esr1 was linearizated by PacI and transfected into HEK293 cells for packaging,amplifying and purifying to obtain recombined adenovirus Ad-Esr1 and its titer measured.HEK293 cell was infected by recombinated adenovirus Ad-Esr1 and Esr1 protein was detected by Western Blot.RESULTS：It was confirmed by sequencing that the extracted CDS fragment of Esr1 gene had no mutation.Restriction endonuclease analysis and sequencing confirmed the successful cloning of the gene into the recombined adenovirus.Ad-Esr1 was successfully constructed with a titer of 6.4×1013（pfu/L）.Western blot showed Esr1 protein expression in infected HEK293 cells and didn＇t express in uninfected HEK293 cells.CONCLUSION：The recombinant adenovirus of Esr1 gene has been successfully constructed,which provides a basis for the research into the role of Esr1 about the effect of biological function in nervous system and gene therapy in nervous system disease.