兔出血症病毒感染性cDNA真核表达载体的构建与鉴定

Construction and identification of eukaryotic vector plasmid of the infectious clone of rabbit hemorrhagic disease virus

【目的】构建操作更简便的兔出血症病毒(RHDV)感染性cDNA真核表达载体。【方法】在前期构建的RHDV感染性克隆基础上,将其全长cDNA分子分为3段,利用单一的限制性内切酶将其依次克隆至真核表达载体pcDNA3.1(+)上,构建RHDV感染性cDNA真核表达质粒,将该质粒转染BHK-21细胞,传3代后,对产生明显细胞病变的细胞冻融液进行RT-PCR检测。【结果】RHDV感染性cDNA真核表达载体构建成功。构建质粒转染的BHK-21细胞出现明显细胞病变,RT-PCR检测结果表明,RHDV拯救成功。【结论】利用真核表达载体可以很方便地拯救出RHDV。

[Objective] The study was done to construct a research platform which is easy to operate the infectious clone of rabbit hemorrhagic disease virus （RHDV）. [Methodl On the basis of pre-built infectious clone of RHDV,the full-length cDNA was divided into three fragments using the restriction endonuclease. The three fragments were ligated to the pcDNA3.1（＋） plasmid in proper order in order to construct the eukaryotic vector of the infectious clone of RHDV. Then, the plasmid was transcripted into BHK-21 cells,the virus was collected and passed on for three generations. Next, the transfected BHK-21 cells which had the significant CPE were detected by RT-PCR. [Result] The new vector was successfully constructed. The significant CPE of transfected BHK-21 cells and RT-PCR amplification indicated that the cDNA clones were rescued successfully in BHK-21 cells. [Conclusion] The pcDNA3.1 （＋） eukaryotic expression vector can rescue the infectious RHDV easily.