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目的获取人嗜酸粒细胞亲合素(Eotaxin)N端突变体(Met/Eotaxin)重组腺病毒,探讨其在小鼠变应性鼻炎中的作用。方法从人外周血单个核细胞总RNA中克隆人400bp的Eotaxin基因;通过N端点突变获取Met/Eotaxin基因,构建pAdtrack-CMV-Met/Eotaxin腺病毒穿梭质粒;将该质粒在大肠杆菌BJ5183中与腺病毒基因组质粒进行同源重组;将Met/Eotaxin重组腺病毒载体在AD293细胞中包装、扩增获取Met/Eotaxin重组腺病毒(Ad-Met/Eotaxin);采用聚合酶链式反应(polymerase chain reaction,PCR)和蛋白印迹杂交(Western blot)对Met/Eotaxin重组腺病毒的正确性进行鉴定。采用卵清蛋白(ovalbumin,OVA)致敏和激发诱导小鼠变应性鼻炎,40只小鼠随机分为变应性鼻炎组和Ad-Met/Eotaxin组。Ad-Met/Eotaxin组在每次激发前30min予以每只小鼠2ml滴度为5×109pfu/ml的Ad-Met/Eotaxin腹腔注射。比较二组动物变应性鼻炎症状和鼻黏膜形态学改变,并在荧光显微镜下观察腺病毒转染效率标志物———绿色荧光蛋白在鼻黏膜等组织中的表达。结果PCR和Western blot结果显示Ad-Met/Eotaxin构建正确;Ad-Met/Eotaxin腹腔注射后可高效转染小鼠鼻黏膜;Ad-Met/Eotaxin可显著减轻小鼠变应性鼻炎症状以及鼻黏膜形态学改变。结论Ad-Met/Eotaxin具有阻断小鼠变应性鼻炎的作用。
Objective To obtain the recombinant adenovirus Eotaxin N-terminal mutant (Met / Eotaxin) and investigate its role in allergic rhinitis in mice. METHODS: A human 400 bp Eotaxin gene was cloned from total RNA of human peripheral blood mononuclear cells. The Met / Eotaxin gene was obtained by N-terminal mutation to construct the shuttle plasmid pAdtrack-CMV-Met / Eotaxin. The plasmid was ligated into E. coli BJ5183 Adenovirus genome plasmid was homologously recombined. Met / Eotaxin recombinant adenovirus vector was packaged in AD293 cells to obtain Met / Eotaxin recombinant adenovirus (Ad-Met / Eotaxin); polymerase chain reaction , PCR) and Western blot were used to identify the correctness of Met / Eotaxin recombinant adenovirus. Allergen and challenge-induced allergic rhinitis in mice was induced by ovalbumin (OVA), and 40 mice were randomly divided into allergic rhinitis group and Ad-Met / Eotaxin group. The Ad-Met / Eotaxin group was intraperitoneally injected with 2 ml of Ad-Met / Eotaxin with a titer of 5 × 10 9 pfu / ml per mouse 30 min before each challenge. The symptoms of allergic rhinitis and morphological changes of nasal mucosa were compared between the two groups. The expression of green fluorescent protein (GFP) in nasal mucosa was observed under a fluorescence microscope. Results Ad-Met / Eotaxin was constructed correctly by PCR and Western blot. Transfection of Ad-Met / Eotaxin into the nasal mucosa of mice by intraperitoneal injection of Ad-Met / Eotaxin could significantly reduce the symptoms of allergic rhinitis and nasal mucosa Morphological changes. Conclusion Ad-Met / Eotaxin has the effect of blocking allergic rhinitis in mice.