Ⅰ型牛疱疹病毒TK^-/gE^-基因双缺失突变株生物学特性

Study on the Characteristics of BHV-1 TK^-/gE^-/EGFP^+ Gene-deleted Mutant Virus Strain

目的构建Ⅰ型牛疱疹病毒(BHV-1)的TK-/gE-双缺失突变株,检测其生物学功能,为治疗牛传染性鼻气管炎提供参考。方法构建了带有EGFP表达盒的BHV-1 TK-/gE-双缺失突变株。通过southern杂交、蛋白质斑点印迹、空斑试验、MDBK细胞增殖实验等技术方法,对重组基因所构成病毒突变株的生物学特性进行鉴定。结果 Southern杂交及蛋白斑点印迹表明,课题组成功构建了带有EGFP表达盒的双缺失突变株;该突变株具有与野生株相当的繁殖力,但TK-/gE-成功缺失,毒力大幅减弱;BHV-1 TK-/gE-遗传稳定,不会返强。结论本项目组成功构建了Ⅰ型牛疱疹病毒(BHV-1)的TK-/gE-双缺失突变株,该缺失株具有良好的安全性和稳定性,为该突变株进一步作为生物安全疫苗和多价病毒载体提供了依据,为预防和治疗牛传染性鼻气管炎提供了技术支持。

Objective To detect the function of I BHV-1 TK-/gE-/ EGFP＋ gene-deleted mutant virus strain,find a new method to prevent infectious bovine rhinotracheitis（IBR）,the charaction of TK-/gE-/ EGFP＋ gene-deleted mutant virus strain was further studied.Methods Southern hybridization,western dot blot and Plaques test were employed in this investment.Results TK gene is deleted and the mutant cannot produce glycoprotein E when gE gene is deleted.The diameter of mutant strain much smaller than that of field strain,the mutant strain grows on MDBK cells and the virus titer can keep 106.0TCID50/0.1 mL for more than 40 hours,which indicated that the mutant strain inherit instantly.Conclusion The gene-deleted mutant virus strain was successful constructed in this study,and the mutant virus strain was stable.This will be useful for the further study of IBR.