梅花鹿α干扰素基因在MDBK细胞中表达及抗BVDV活性检测

Expression of Cervus nippon interferon alpha gene in MDBK cells and its antiBVDV activity detection

采用基因工程技术克隆出梅花鹿α干扰素(IFN-α)成熟肽基因序列,并将其定向插入到真核表达载体pcDNA3.0(+),经酶切、PCR及测序鉴定,成功构建了pcDNA3.0-IFN-α真核重组表达质粒。将鉴定正确的阳性重组质粒经非脂质体法转染至MDBK细胞,间接免疫荧光试验检测其具有较高转染效率;Western blot法检测IFN-α蛋白在MDBK细胞中得到有效表达;病变抑制试验检测转染后的MDBK细胞具有明显抗BVDV活性;抗BVDV活性的定量试验表明转染pcDNA3.0-IFN-α质粒的MDBK细胞与感染未转染的MDBK细胞相比抗BVDV活力提高了66192倍。本试验在MDBK细胞中成功表达了梅花鹿IFN-α,并检测出其具有明显抗BVDV作用,这为梅花鹿IFN-α活性机理研究以及开发更高效的抗梅花鹿病毒性疾病干扰素制剂提供物质和理论基础。

Genetic engineering technology was adopted to clone the Cervus nippon interferon alpha （IFN-α） mature peptide gene and inserted the gene into eukaryotic expression vector pcDNA3.0 （＋） and the recombinant expression plasmid of pcDNA3.0-IFN-α was successfully constructed by enzyme digestion,PCR and DNA sequencing identification. The positive plasmid was transfected into MDBK cells by non-liposome method of FuGENE~ 6 Transfection Reagent. The transfection efficiency detected by indirect immunofluorescence assay （IFA） was high. Western blot was used to detect IFN-α protein obtained effective expression in MDBK cells. Cytopathic inhibition test was used to detection of anti BVDV activity of transfected MDBK cells,the mensurable examination of anti-BVDV activity showed that in MDBK cells transfected with pcDNA3.0-IFN-α plasmid anti- BVDV activity increased by 66 192 times compared with untransfected normal MDBK cells. The Cervus nippon＇s IFN-α was successfully expression in MDBK cells and it has obviously antiviral activity. These results will provide material and theoretical foundation of Cervus nippon IFN-α s ac- tivity mechanism research and developing more efficient interferon preparations of antiviral disease of Cervus nippon.