表达前强啡肽基因的Ad5型腺病毒载体的构建及鉴定

Construction and identification of recombinant adenovirus serotype 5 vector with prodynorphin gene

目的构建且鉴定含前强啡肽基因（prodynorphin）的血清5型腺病毒载体（Ad5）。方法应用分子生物学方法将前强啡肽基因序列克隆入腺病毒穿梭质粒pDC316-PDP，后者与骨架质粒共转染HEK293细胞，包装得到含前强啡肽转基因的腺病毒Ad5-PDP。用聚合酶链反应（PCR）方法对转基因病毒进行鉴定，TCID50法测定病毒滴度。结果PCR法证实转基因正确插入了Ad5型病毒基因组内，且没有野生型病毒污染，病毒滴度为1×10^12 v．p．／mL。PCR鉴定Ad5-PDP重组成功。结论获得的Ad5-PDP滴度高，感染性好，可以用于转基因治疗的实验研究。

Objective To construct and identify the recombinant adenovirus serotype 5 vector with prodynorphin （PDP） gene. Methods The prodynorphin gene was cloned into the shuttle plasmid pDC316-PDP with the method of molecular biology. The shuttle plasmid pDC316-PDP and genomic plasmids were co-transfected into HEK293 to package the adenovirus Ad5-PDP. The transgenic virus was identified with the method of PCR. The titer of proliferated virus, after purified, was determined by TCID50. Resuits It was identified that the sequence of prodynorphin gene was correctly inserted into the genome of transgenic virus, and not contaminated by wild type virus. The titer of Ad5-PDP was 1×10^12v. p./mL after purification. The identification of PCR and immunocytochemical stain showed that the construction of the recombinant Ad5 -PDP plasmid could be confirmed. Conclusion This Ad5-PDP virus vector with high titer and strong infectivity can be used in empirical study of transgenic therapy.