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表达抑制山羊痘病毒ORF095 shRNA的山羊成纤维细胞系的建立
引用本文:赵志荀,吴国华,颜新敏,许丹,李健,朱海霞,崔力凡,高顺平,孙晓林,马保华,张强.表达抑制山羊痘病毒ORF095 shRNA的山羊成纤维细胞系的建立[J].甘肃农业大学学报,2012(2):6-11.
作者姓名:赵志荀  吴国华  颜新敏  许丹  李健  朱海霞  崔力凡  高顺平  孙晓林  马保华  张强
作者单位:甘肃农业大学动物医学院;中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室;西北农林科技大学动物医学院,农业部动物生理生殖和胚胎工程重点开放实验室
基金项目:农业部转基因生物新品种培育重大专项(2009ZX08008-010B);国家自然基金(31001056)
摘    要: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.

关 键 词:山羊痘病毒  pGPU6/GFP-shRNA  G418  细胞系  RT-PCR

Constructing fiber cell lines integrated with shRNA targeted ORF095 of GTPV
ZHAO Zhi-xun,WU Guo-hua,YAN Xin-min,XU Dan,LI Jian,ZHU Hai-xia,CUI Li-fan,GAO Shun-ping,SUN Xiao-lin,MA Bao-hua,ZHANG Qiang.Constructing fiber cell lines integrated with shRNA targeted ORF095 of GTPV[J].Journal of Gansu Agricultural University,2012(2):6-11.
Authors:ZHAO Zhi-xun  WU Guo-hua  YAN Xin-min  XU Dan  LI Jian  ZHU Hai-xia  CUI Li-fan  GAO Shun-ping  SUN Xiao-lin  MA Bao-hua  ZHANG Qiang
Institution:1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;2.Key Laboratory of Animal Virology of the Ministry of Agriculture,State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agriculture Sciences,Lanzhou 730046,China;3.Key Laboratory of Animal Reproductive Physiology & Embryo Technology,Ministry of Agriculture,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)
Abstract: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.
Keywords:goatpox virus  pGPU6/GFP-shRNA  G418  cell line  RT-PCR
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