猪圆环病毒2型orf2基因在奶山羊胎儿成纤维细胞中的表达与生物活性鉴定

Expression of porcine circovirus virus type 2 orf2 gene in fetal fibroblasts of dairy goats and identification of its biological activity

为获得稳定表达猪圆环病毒2型(PCV-2)orf2基因的奶山羊胎儿成纤维细胞,将PCV-2 orf2基因与筛选基因neo表达框串联插入到pBC1质粒,得到重组的真核表达载体pBC1-orf2-neo。然后采用脂质体介导法将重组质粒转染至奶山羊胎儿成纤维细胞(gFFs),经G418筛选获得具有稳定抗性的阳性gFFs细胞株。RT-PCR分析显示,在获得的阳性gFFs细胞株中能够扩增出689bp的orf2基因和1 953bp的neo表达框,与预期结果相符。Western blot分析显示,获得了约37 000的表达产物,且能够与兔抗PCV-2Cap蛋白抗体发生反应。结果表明,orf2基因成功整合到山羊胎儿成纤维细胞染色体上并能够正确表达,表达产物具有良好的反应原性,这为构建分泌Cap蛋白的奶山羊乳腺生物反应器奠定了理论和物质基础。

To obtain the goat fetal fibroblasts stably expressing porcine circovirus virus type 2 （PCV-2）orf2, the PCV-2 gene or f2 and neo expression cassette were cloned into pBC1 vector, termed pBCl-orf2-neo. Prepared plasmids were transfected into the dairy goat fetal fibroblasts （gFF） cells. Stably resistant gFF cells were obtained after G418 screening. RT-PCR analysis indi- cated that both the 689 bp off2 gene and 1 953 bp neo expression cassette were present in gFF cells. Western blot results showed that the predicted 37 000 product was obtained and the product reacts to rabbit anti PCV-2 antibody. These work showed that the off2 gene was successfully inte- grated into the chromosomes of goat fetal fibroblast cells,and the gene product conducted good re- actionogenicity like PCV-2 Cap protein. Our work laid both theoretical and practice foundation for constructing the dairy goat mammary gland bioreactor for Cap protein.