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

发布时间：2018-04-20 05:43

本文选题：猪圆环病毒 + 乳腺表达载体　；参考：《中国兽医学报》2017年09期

【摘要】：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。
[Abstract]:In order to obtain goat fetal fibroblasts stably expressing porcine circovirus type 2 PCV-2 orf2 gene, the PCV-2 orf2 gene and the screening gene neo expression frame were inserted into the pBC1 plasmid in tandem to obtain the recombinant eukaryotic expression vector pBC1-orf2-neo. Then the recombinant plasmid was transfected into dairy goat fetal fibroblasts by liposome-mediated transfection. Positive gFFs cell lines with stable resistance were obtained by G418 screening. RT-PCR analysis showed that the recombinant plasmid had stable resistance. The orf2 gene of 689bp and the neo expression box of 1 953bp could be amplified from the positive gFFs cell line. Western blot analysis showed that about 37,000 expression products were obtained and could react with rabbit anti- protein antibody. The results showed that the gene of orf2 was successfully integrated into the chromosomes of goat fetal fibroblasts and could be expressed correctly. This laid a theoretical and material foundation for the construction of milk goat mammary gland bioreactor secreting Cap protein.
【作者单位】：甘肃农业大学生命科学技术学院;甘肃农业大学动物医学院;河南省农业科学院动物免疫学重点实验室;河南农业大学牧医工程学院;
【基金】：兰州市科技局人才创新创业资助项目(2015-RC-7) 河南省农业科学院动物免疫学重点实验室专项资助项目
【分类号】：S852.651

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