输卵管特异表达人溶菌酶基因重组禽腺联病毒的构建及鉴定

发布时间：2018-10-08 19:50

【摘要】：试验旨在利用杆状病毒表达系统制备输卵管特异表达人溶菌酶(h LYZ)的重组禽腺联病毒(recombinant avian adeno-associated virus,r AAAV)。参照已发表的h LYZ基因序列设计1对引物,PCR扩增h LYZ基因片段,亚克隆至含输卵管特异表达盒和禽腺联病毒(AAAV)两侧末端反向重复序列(inverted terminal repeat,ITR)的转移载体中,获得重组杆状病毒转移载体p FB-AIOVLYZ,将其转化到大肠杆菌DH10Bac感受态细胞中,经抗性和蓝白斑筛选,获得重组穿梭质粒r Bacmid-AIOVLYZ,在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒r Bac-AIOVLYZ。将其与表达AAAV结构蛋白的重组杆状病毒r Bac-VP及表达AAAV功能蛋白的重组杆状病毒r Bac-Rep以感染复数(multiplicity of infection,MOI)为5感染Sf9昆虫细胞,72 h后收集细胞沉淀,并经滤膜过滤、氯仿抽提和PEG沉淀,即为r AAAV-OVLYZ。电镜结果显示,病毒粒子大小约为20 nm,形态结构与野生AAAV相似;PCR结果显示r AAAV含有目的基因;体外细胞表达试验说明r AAAV能介导h LYZ在输卵管细胞中的特异表达。结果表明,本研究利用杆状表达系统成功制备了输卵管特异表达h LYZ基因的r AAAV,为h LYZ的大量制备奠定了基础。
[Abstract]:The aim of the experiment was to prepare the recombinant avian adenosine virus (recombinant avian adeno-associated virus,r AAAV).) expressing human lysozyme (h LYZ) by using baculovirus expression system. According to the published h LYZ gene sequence, a pair of primers were designed to amplify the h LYZ gene fragment and subcloned into the transfer vector containing the oviduct specific expression box and the reverse repeat sequence (inverted terminal repeat,ITR at the bilateral end of Avian adenovirus (AAAV). The recombinant baculovirus transfer vector p FB-AIOVLYZ, was obtained and transformed into Escherichia coli DH10Bac competent cells. After screening with resistance and blue and white spot, the recombinant shuttle plasmid r Bacmid-AIOVLYZ, was transfected into Sf9 insect cells mediated by liposome, and the recombinant baculovirus r Bac-AIOVLYZ. was obtained. The recombinant baculovirus r Bac-VP expressing AAAV structural protein and the recombinant baculovirus r Bac-Rep expressing AAAV functional protein were infected with (multiplicity of infection,MOI for 72 h. Chloroform extraction and PEG precipitation, i.e. r AAAV-OVLYZ. The results of electron microscopy showed that the size of the virus particles was about 20 nm, which was similar to that of wild AAAV. The results showed that r AAAV contained the target gene, and that r AAAV could mediate the specific expression of h LYZ in oviduct cells in vitro. The results showed that the r AAAV, which specifically expressed the h LYZ gene was successfully prepared by using the rod expression system in this study, which laid a foundation for the preparation of h LYZ.
【作者单位】：江苏农牧科技职业学院江苏省兽用生物制药高技术研究重点实验室;
【基金】：国家自然科学基金项目(31302096) 江苏省农业支撑项目(BE2013415) 江苏省六大人才高峰项目(NY-009)
【分类号】：Q78

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