中国寄生虫学与寄生虫病杂志 ›› 2006, Vol. 24 ›› Issue (5): 9-359.

• 实验研究 • 上一篇    下一篇

柔嫩艾美球虫杂交株F2 SO7基因重组鸡痘病毒的构建和筛选

杨桂连1,2,张西臣1;赵权2;李建华1;尹继刚1   

  1. 1吉林大学畜牧兽医学院, 长春 130062; 2 吉林农业大学动物科学技术学院, 长春 130118
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-10-30 发布日期:2006-10-30
  • 通讯作者: 张西臣

Construction and Screening of Recombinant Fowlpox Virus ExpressingEimeria tenella F2 Hybrid Strain SO7 Gene

YANG Gui-lian1,2;ZHANG Xi-chen1;ZHAO Quan2;LI Jian-hua1;YIN Ji-gang1   

  1. 1 College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China;2 College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2006-10-30 Published:2006-10-30
  • Contact: ZHANG Xi-chen

摘要: 目的 构建柔嫩艾美球虫(E.tenella)杂交株F2 SO7基因重组鸡痘病毒。 方法 将柔嫩艾美球虫杂交株F2 SO7基因,插入到以胸苷激酶(TK)基因为侧翼的鸡痘病毒表达载体pUTA2中的复合启动子下游,获得重组表达质粒pUTA-SO7。用脂质体将重组表达质粒转染鸡痘病毒(FPV)感染的鸡胚成纤维细胞(CEF),培养、收获病毒后,用含40 mg/L 5-溴-2-脱氧尿嘧啶(BrdU)的培养液,在TK基因阳性CEF细胞中筛选培养2代,然后用不含BrdU的培养液进行病毒噬斑纯化以筛选rFPV。 结果 PCR扩增可见650 bp左右蛋白条带,间接荧光抗体试验(IFAT)可见重组病毒感染细胞表面有绿色荧光物质,蛋白质印迹分析(Western Blotting),在相对分子质量(Mr)36 000处有1条特异条带,证实了重组病毒在CEF中表达了SO7基因。 结论 成功筛选出表达E.tenella杂交株F2 SO7基因的重组鸡痘病毒。

关键词: 柔嫩艾美球虫, SO7, 重组, 鸡痘病毒

Abstract: Objective To Construct the recombinant fowlpox virus expressing Eimeria tenella F2 hybrid strain SO7 gene. Methods A recombinant expression plasmid pUTA-SO7 was constructed by inserting the SO7 gene of Eimeria tenella F2 hybrid strain into downstream of a hybrid poxvirus promoter which was flanked by the TK gene of fowlpox virus(FPV). The constructed pUTA-SO7 was firstly transfected into chicken embryo fibroblast cells(CEF) pre-infected with FPV strain 282E4 by using liposome, then the viruses resulted from the transfection were selected for 2 passages by culturing in CEF cells with MEM medium containing 40 mg/L 5-bromo-2-deoxy-uridine(BrdU). The selected viruses were plaque-purified in CEF cultured with MEM medium without BrdU. Results Polymerase chain reaction (PCR), indirect immunofluorescence assay and Western blotting showed that SO7 gene was expressed in recombinant fowlpox virus. Conclusion The recom-binant FPV (rFPV) expressing the SO7 gene has been obtained.

Key words: Eimeria tenella, SO7, Recombinant, Fowlpox virus