表达重组鸡新城疫病毒免疫原蛋白基因的研究

STUDY ON EXPRESSION OF NEWCASTLE DISEASE VIRUS IMMUNOPROTEIN

以 Ｎ Ｄ Ｖ Ｈ Ｎ 基因重组禽痘病毒载体与禽痘病毒转染液为材料，以载体质粒的 Ｌａｃ Ｚ报告基因为筛选标记，在含有 Ｘｇａｌ的培养基上筛选蓝色空斑的阳性重组子，对初次筛选到的蓝色空斑经过三轮蚀斑纯化（蓝斑形成率达９０％ 以上），得到了能稳定增殖的病毒子；再分别提取病毒子感染细胞、正常对照细胞基因组 Ｄ Ｎ Ａ， Ｂａｍ ＨⅠ酶切后，以 Ｄ Ｉ Ｇ 标记的探针进行 Ｓｏｕｔｈｅｒｎ 杂交检测，发现重组病毒感染的细胞基因组 Ｄ Ｎ Ａ 在７ ｋｂ 处有一 Ｄ Ｎ Ａ 片段，能与 Ｈ Ｎ 特异性探针呈阳性反应，而对照细胞没有，从而利用 Ｌａｃ Ｚ报告基因、 Ｈ Ｎ 探针杂交检测，我们成功地筛选并获得了 Ｎ Ｄ Ｖ Ｈ Ｎ 基因重组禽痘病毒，为下一步的研究工作奠定了基础。

In the study,by using report gene LacZ,positive recombinant virus was screened in the cotransfection culture fluid of recombinant fowl pox vector with the wild fowl pox virus.After three cycles of blue plaque purification,the plaque formation rate was over 90%.Furthermore,the genome DNAs of chicken embryo cells noninfected and cells infected with recombinant virus were extracted and digested by BamHⅠ for Southern blot.In comparison with CEF,there is a DNA fragment in the genome of the infected cells and it can react with anti NDV HN antibodies.Thus by three cycles of plaque purification and Southern blotting,the positive recombinant fowl pox virus can be available.