禽腺联病毒序列对鸡输卵管细胞表达人溶菌酶基因的促进作用

Enhancement of avian adeno-associated virus sequence on expression of human lysozyme gene in oviduct cells

将鸡卵清蛋白基因5′-调控区控制的人溶菌酶(hLYZ)cDNA表达盒插入禽AAV末端反向重复序列之间,将CMV启动子控制的rep表达盒插入其下游,获得的表达载体pRep2ITR-OV4-LYZ用聚乙烯亚胺包裹,经翅静脉注射产蛋鸡,待蛋清中rhLYZ表达水平下降至注射前水平时,用pRep2ITR-OV4-LYZ进行3次重复注射,以RBB-R染料标记比色法和Western-blotting检测蛋清中rhLYZ表达,以细菌生长抑制试验检测rhLYZ活性。结果,在相同试验条件下,pRep2ITR-OV4-LYZ表达rhLYZ的水平和时间优于不含禽AAV序列的pOV4-LYZ,rhLYZ表达水平和时间随载体注射次数增加呈上升和延长趋势,rhLYZ的分子质量与天然hLYZ相同,对致乳牛乳腺炎大肠杆菌、葡萄球菌和链球菌具有显著的抑制作用。试验结果提示,禽AAV ITR和rep序列能促进hLYZ基因在鸡输卵管细胞中正确表达。

Human lysozyme（hLYZ） cDNA expression cassette control led by 5＇-regulatory region of ovalbumin gene was flanked by avian adeno-associated virus（AAV） inverted terminal repeats（ITR） and rep-expression cassette control led by CMV promoter was included in the same vector, resulting in a new oviduct-specific expression vector pRep2ITR-OV4-LYZ. After mixing with polyethylen imine, 1 × 10^14 copies of vector pRep2ITR-OV4-LYZ or control vector pOV4-LYZ were injected into egg-laying hens via wing vein and repeated injections were performed two weeks later. The egg whites were collected for enzymatic assay and Western-blotting. The expression of rhLYZ gene was significantly higher and longer in the egg whites of pRep2ITR-OV4-LYZ-injected hens than that of pOV4-LYZ-injected birds,and longer expression was obtained by repeated injection. Western-blotting showed that the expressed rhLYZ had the same molecular weight with that of natural hLYZ. In vitro bacterial growth inhibition tests showed that rhLYZ significantly inhibited the growth of cow mastitis-caused by Escherichia coli, Staphylococcus and Streptococcus. These results indicated that avian AAV ITRs and rep-coding sequence were able to promote hLYZ gene＇s expression in oviduct cells.