中文摘要：

目的：构建表达虫荧光色素酶报告基因重组腺病毒载体,为腺病毒中和抗体流行水平的定量检测奠定基础。方法：采用腺病毒表达系统（ViraPower Adenoviral Expression System）构建重组腺病毒表达载体。首先利用PCR的方法扩增虫荧光色素酶基因使其具备特定的CACC接头,以连接、转化、提取质粒等方法克隆入载体pENTR/D-TOPO以获得入门克隆,经PCR及测序鉴定正确后,用重组酶（LR ClonaseTM Ⅱ Enzyme Mix）进行入门克隆与表达载体（pAd-CMV/V5-DEST）间的重组反应,以获得表达克隆rAd-Luci。表达克隆鉴定后,用限制性内切酶PacⅠ线性化后转染HEK293A包装细胞得到重组腺病毒。经过扩增后,用极限稀释法检测病毒滴度,用Western blot法检测rAd-Luci载体是否能正确表达虫荧光色素酶蛋白,并检测此酶蛋白的功能活性。结果：用PCR的方法扩增到具有CACC接头的虫荧光色素酶基因,其重组入门和腺病毒表达克隆经PCR和测序鉴定构建正确,重组表达克隆转染HEK293A细胞并扩增后获得的病毒滴度为1.8×1011 pfu/ml,此病毒能正确表达虫荧光色素酶蛋白,并且具有较强的功能活性。结论：成功构建了虫荧光色素酶报告基因重组腺病毒载体,为此重组载体用于腺病毒中和抗体的定量检测奠定基础。

英文摘要：

Objective：In order to lay the foundation for quantitative detection of adenovirus neutralizing antibodies,a luciferase gene recombinant adenovirus vector is aimed to be constructed.Methods：The adenoviral expression system,ViraPowerTM Adenoviral Expression System,was used to construct the recombinant adenovirus vector.Using PCR method,luciferase gene was amplified from the pGL3 plasmid and CACC was added at the 5 end.After ligation and transformation,the luciferase gene was cloned into the entry vector,pENTR/D-TOPO,and identified by sequencing and PCR.LR ClonaseTM Ⅱ Enzyme Mix was used to recombine entry-cloning with recombinant expression vector（pAd-CMV/V5-DEST）to generate expression vector plasmid,rAd-Luci.After identification by PCR,the rAd-Luci plasmid was linearized by the restriction enzymes,PacⅠ,and transfected HEK293A packaging cells.After amplification of the recombinant adenovirus,limiting dilution method was used to detect the virus titer.Western blot was used to detect the expression of luciferase protein.Results：The luciferase gene was obtained by PCR method.The adenoviral entry and expression clones were identified correctly by PCR and sequencing.Those correct recombinant adenoviral expression plasmids transfected HEK293A cells to obtain the recombinant adenovirus with virus titer of 1.8×1011 pfu/ml.This recombinant virus was able to express functional luciferase protein correctly.Conclusion：The construction of luciferase gene recombinant adenovirus vector was successful and this study has laid an experimental basis for quantitative detection of adenovirus neutralizing antibodies.