| 摘要: |
| 【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。 |
| 关键词: 兔出血症病毒感染性cDNA pcDNA3.1(+) BHK-21细胞 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目(30800045，30670074，30870114);浙江省自然科学基金项目“家畜疫病病原生物学国家重点实验室开放基金”(Y305047) |
|
| Construction and identification of eukaryotic vector plasmid of the infectious clone of rabbit hemorrhagic disease virus |
|
|
| Abstract: |
| 【Objective】 The study was done to construct a research platform which is easy to operate the infectious clone of rabbit hemorrhagic disease virus (RHDV).【Method】 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. |
| Key words: infectious clone of rabbit hemorrhagic disease virus (RHDV) pcDNA3.1（＋） BHK-21 cell |
