[表达柔嫩艾美耳球虫F2杂交株SO7基因的重组鸡痘病毒的构建与筛选]

[Construction and screening of recombinant fowlpox virus expressing Eimeria tenella F2 hybrid strain SO7 gene].

作者信息

Yang Gui-lian, Zhang Xi-chen, Zhao Quan, Li Jian-hua, Yin Ji-gang

机构信息

College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China.

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006 Oct;24(5):356-9.

PMID:17361816

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 S07 gene was expressed in recombinant fowlpox virus.

CONCLUSION

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

摘要

目的

构建表达柔嫩艾美耳球虫F2杂交株SO7基因的重组鸡痘病毒。

方法

将柔嫩艾美耳球虫F2杂交株的SO7基因插入到由鸡痘病毒（FPV）TK基因侧翼的杂交痘病毒启动子下游，构建重组表达质粒pUTA-SO7。利用脂质体将构建好的pUTA-SO7首先转染至预先感染了FPV 282E4株的鸡胚成纤维细胞（CEF）中，然后将转染后产生的病毒在含有40 mg/L 5-溴-2-脱氧尿苷（BrdU）的MEM培养基中培养的CEF细胞中进行2代筛选。将筛选出的病毒在不含BrdU的MEM培养基中培养的CEF中进行空斑纯化。结果聚合酶链反应（PCR）、间接免疫荧光试验和蛋白质印迹法表明SO7基因在重组鸡痘病毒中表达。