柔嫩艾美球虫杂交株F2 SO_7基因重组鸡痘病毒的构建和筛选

Construction and Screening of Recombinant Fowlpox Virus Expressing Eimeria tenella F2 Hybrid Strain SO_7 Gene

目的构建柔嫩艾美球虫(E.tenella)杂交株F2SO7基因重组鸡痘病毒。方法将柔嫩艾美球虫杂交株F2SO7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40mg/L5-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650bp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(WesternBlotting),在相对分子质量(Mr)36000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F2SO7基因的重组鸡痘病毒。

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 S07 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 recombinant FPV （rFPV） expressing the SO7 gene has been obtained.