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

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（hLYZ）的重组禽腺联病毒（recombinant avian adeno-associated virus，rAAAV）。参照已发表的hLYZ基因序列设计1对引物，PCR扩增hLYZ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pFB-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rBacmid-AIOVLYZ，在脂质体介导下转染Sf9昆虫细胞，获得重组杆状病毒rBac-AIOVLYZ。将其与表达AAAV结构蛋白的重组杆状病毒rBac-VP及表达AAAV功能蛋白的重组杆状病毒rBac-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rAAAV-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rAAAV含有目的基因；体外细胞表达试验说明rAAAV能介导hLYZ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hLYZ基因的rAAAV，为hLYZ的大量制备奠定了基础。

Construction and Identification of Recombinant Avian Adeno-associated Virus Oviduct-specific Expressing Human Lysozyme Gene

In order to produce recombinant avian adeno-associated virus (rAAAV) oviduct-specific expressing human lysozyme (hLYZ) by the baculovirus expression system, one pair of primers was designed according to the published sequences for hLYZ, the hLYZ gene was amplified by PCR and cloned into baculovirus expression vector, which contained the oviduct-specific expression cassette and the inverted terminal repeats of avian adeno-associated virus (AAAV), the resulted plasmid was named as pFB-AIOVLYZ.Then the recombinant vector pFB-AIOVLYZ was transformed into E.coli DH10Bac, and the positive recombinant bacmid rBacmid-AIOVLYZ was screened according to the resistant and the blue-white plague screening,rBacmid-AIOVLYZ was transfected into the Sf9 insect cells by liposome. Once the cytopathic effect was found, the rBac-AIOVLYZ could be harvested.Sf9 insect cells cultured in suspension culture were infected with three recombinant baculoviruses, rBac-AIOVLYZ, rBac-VP and rBac-Rep, at an MOI of 5. 72 h later, Sf9 insect cells were collected, and recombinant viral particles rAAAV-OVLYZ were purified by filtration, chloroform extraction and PEG precipitation. Electron microscopy showed a typical morphologic feature of Parvoviridae family with virus particle size of about 20 nm. PCR results indicated that the target gene existed in the viral genome. The in vitro cell expression test showed that rAAAV could mediate the specific expression of hLYZ in oviduct cells. These results indicated that the rAAAV oviduct-specific expressing hLYZ was successfully prepared by baculovirus expression system, which laid the foundation for the preparation of hLYZ.