SV40LT基因重组腺病毒载体的包装、鉴定及用于转染日本血吸虫童虫细胞的研究

Package of recombinant adenovirus vector carrying with SV40LT gene and its expression in Schistosomula cells

目的构建携带SV40LT基因的重组腺病毒表达载体,制备具有感染力的重组腺病毒,观察其转染日本血吸虫(Schistosoma japonicum,Sj)童虫细胞后的表达情况。方法采用体外连接法构建好的携带SV40LT基因的重组腺病毒质粒(AdHu5-SV40LT)转化Stbl2感受态菌,获得重组腺病毒质粒后,经Pac I酶切线性化后转染293A细胞,获得出重组腺病毒(AdHu5-SV40LT)。将重组腺病毒感染Sj童虫细胞,采用RT-PCR和免疫组织化学检测SV40LT基因的表达情况。结果重组腺病毒载体质粒可转染293A细胞并可在293A细胞内进行有效的复制;提取病毒DNA进行PCR检测证实含有SV40LT目的基因。以重组腺病毒能感染Sj童虫细胞,经RT-PCR和免疫组化检测有SV40LT基因在细胞中的表达。结论成功包装了具有感染能力的含SV40LT基因的重组腺病毒,感染Sj童虫细胞后目的基因有表达,为进一步探索日本血吸虫细胞永生化提供了实验依据。

In order to pack the recombinant adenovirus vector carrying SV40LT gene and to study the effect of the recombinant adenovirus on transfecting schistosomulum cells,The constructed recombinant adenovirus plasmid was re-transformed into competent cells Stbl2,and identified by restriction enzyme digestion.The right recombinant adenovirus plasmid was transfected into 293A cells using Lipofectine 2000.The recombinant adenovirus was amplified,and then identified by polymerase chain reaction（PCR）.The effect of the recombinant adenovirus transfecting schistosomulum cells was detected by RT-PCR and immunohistochemistry.The recombinant adenovirus containing SV40LT gene was successfully packed,which effectively infected schistosomulum cells.The expression of SV40LT gene in schistosomulum cells was detected by RT-PCR and immunohistochemistry.The recombinant adenovirus containing SV40LT gene could transfect schistosomulum cells to provide a basis for Schistosoma japonicum cell immortalization.