表达抑制山羊痘病毒ORF095 shRNA的山羊成纤维细胞系的建立

Constructing fiber cell lines integrated with shRNA targeted ORF095 of GTPV

将已经通过体外验证能够有效抑制山羊痘病毒复制的shRNA转入山羊体细胞,构建其表达细胞系.将已经通过验证能够靶向抑制山羊痘病毒ORF095基因并有效抑制GTPV的pGPU6/GFP-ORF095-siRNA-70转染山羊原代成纤维细胞,经800μg/mL G418抗性筛选7～8d后,用含200μg/mL G418和10～15ng/mL EGF的培养基进一步单克隆化并将单克隆细胞扩大、传代培养,应用RT-PCR检测及测序鉴定所构建的细胞系.结果表明:本试验获得的1株成纤维细胞系基因组含有ORF095-siRNA-70表达框架,为进一步进行体细胞克隆转基因动物的制备和RNAi体内抗山羊痘病毒的研究奠定了基础.

shRNA which could effectively knocked off the goatpox virus was transfected into goat fiber cell, and expression of somatic cells line was constructed, pGPU6/GFP-ORF095-siRNA-70 had been validated that it could inhibit GTPV replication targeted ORF095 gene and effectively restrain the GTPV. Original generation fibroblasts from ＇XiNong milk goat ＇ were transfected with the plasmids, and after 7 to 8 days resistance with 800μg/mL G418,medium containing with 200 μg/mL G418 and 10--15 ng/mL EGF was used to screening of monoclonal cells and further monoclonal cultivation. RT-PCR and sequencing were used to detect the cell lines obtained. The results showed that the experiment got one cell lines genome contained ORF095-siRNA-70 expression framework.