表达人B7-1的重组腺病毒的制备及初步鉴定

目的构建表达B7-1基因的重组腺病毒载体,制备相应的复制缺陷型重组腺病毒并检测其在喉癌细胞中的表达。方法构建携带人B7-1基因的重组穿梭载体pAdtrack-B7-1,PmeI线性化后与腺病毒载体pAdEasy-1共转化大肠杆菌BJ5183,通过同源重组得到重组腺病毒载体pAd-B7-1。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒,应用病毒悬液感染人喉癌细胞株,RT-PCR检测感染细胞中B7-1基因mRNA表达。结果酶切鉴定证实阳性pAdTrackB7-1重组穿梭载体含有目的基因B7-1,同源重组后制备的病毒载体DNA经酶切鉴定获得了阳性重组腺病毒载体,该载体能有效转染HEK293细胞并在细胞内成功包装,转染3d后可以观察到绿色荧光蛋白(GFP)表达并逐渐增多、增强。制备的Ad-B7在体外能有效感染Hep-2细胞,感染后可维持6d高水平的B7-1的表达,整体表达可持续两周。结论成功构建了同时表达B7-1的重组腺病毒载体并制备出重组腺病毒颗粒,该病毒能有效地感染喉癌细胞并表达高水平的B7-1基因,为进一步研究B7-1的功能及应用B7-1进行基因治疗奠定基础。

Construction and identification of recombinant adenovirus expressing B7-1 genes

Objective To construct replication deficient recombinant adenovirus expressing b7-1 genes using AdEasy system, and detect the expression of b7-1 genes in Hep-2 after adenovirus infection. Methods A recombinant adenovirus shutter plasmid pAdTrackB7-1 containing human B7-1 gene was constructed by molecular cloning. The recombinant shutter plasmid was lined by Pme I and co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for homologous recombination to obtain the recombinant adenoviral vector pAd-B7-1. The recombinant adenoviral vector was then transfected into HEK293 package cells to produce virus particles. The expression of B7-1 gene in cells were detected in human laryngeal epithelial carcinoma cells Hep- 2 by RT-PCR after adenovirus infection. Results The recombinant adenoviral vector was successfully established and confirmed by restriction endonuclease digestion. GFP expression was observed on the 3rd day after the adenovirus transfecting into HEK 293 cells. Recombinant adenovirus was identified by PCR. High level Expression of B7-1 mRNA was maintained for 6 days in Hep-2 cells after adenovirus infection. Conclusion The construction of recombinant adenovirus of Ad-B7-1 provided a basis for further studies of combined gene therapy for laryngeal cancers.