以具有WPRE调控元件的杆状病毒为载体在鸡胚原代细胞中表达新城疫病毒F基因  

Construction of baculovirus vector with WPRE regulatory element to express Newcastle disease virus F gene in primary chicken embryo cells

作　　者：高冬妮[1] 平文祥[1] 金丽颖[1] 沈万力 宋刚[1] 唐晓艳[1] 安琦[1] 申燕[1] 葛菁萍[1] 

机构地区：[1]微生物黑龙江省高校重点实验室 黑龙江大学生命科学学院,黑龙江哈尔滨150080

出　　处：《微生物学报》2014年第4期455-462,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31270143);黑龙江大学高层次人才(创新团队)支持计划(Hdtd2010-17);黑龙江省教育厅科学技术研究项目(12511423);黑龙江省高等学校科技创新团队(农业微生物发酵技术)~~

摘　　要：【目的】构建具有哺乳动物细胞特异性启动子和基因转录后调控元件的重组杆状病毒转移载体,优化的重组杆状病毒可介导新城疫病毒(Newcastle disease virus,NDV)F基因在鸡原代骨骼肌细胞中进行高效表达。【方法】提取NDV La Sota株病毒RNA基因组,用RT-PCR技术扩增F基因,将其克隆到自主构建的具有转录后调控元件(Woodchuck hepatitis virus post-transcriptional regulatory element,WPRE)的杆状病毒转移载体的CMV启动子下游,通过Bac-to-Bac系统获得F重组Bacmid,经转染Sf9昆虫细胞后获得F重组杆状病毒。重组病毒经扩增后以50个MOI感染鸡原代骨骼肌细胞,接种72 h后裂解细胞收获蛋白。比较分析WPRE调控元件对蛋白表达水平的影响。【结果】经Western blot证实在鸡原代骨骼肌细胞中表达了NDV F蛋白,分子量约56 kDa,与预测蛋白大小一致,且能被NDV阳性血清所识别。添加WPRE转录后调控元件的重组杆状病毒介导F蛋白的表达效果与添加10 mmol/L丁酸盐法基本相同,但对细胞几乎没有毒性。【结论】优化后的重组杆状病毒可以向鸡原代细胞中传递NDV F基因,并在CMV的启动下表达具有反应原性的F蛋白,添加的转录后调控元件WPRE可以增强杆状病毒介导外源基因在鸡原代细胞中的表达水平。本研究为研制NDV及其他重要禽类传染病的杆状病毒载体基因工程疫苗奠定了基础。[ Objective] To construct the recombinant baculovirus with mammaliancell-specific promoter and woodchuck hepatitis virus post-transcriptional regulatory element （WPRE）, to highly express Newcastle disease virus（NDV） F gene in the primary chicken embryo ceils. [Method] We extracted total RNAs from NDV La Sota strain. Then the F gene was amplified by reverse transcription polymerase chain reaction. We constructed the baculoviral vector （pCMV-WPRE-F） with F gene fused with the WPRE near its 3＇end, which expressed under the control of the CMV promoter. The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus. After amplification of recombinant baculovirus, the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection, and the proteins were levels mediated by WPRE regulatory element were analyzed harvested at 72 h after infection. The F protein expression [ Results] Western blot results show that the F gene was successfully expressed in chicken primary cells. The product was a 56kDa protein and could be recognized by anti-NDV serum. The WPRE fusion significantly improved the F gene expression as 10mmol/L butyrate did, but different to butyrate, the WPRE regulatory element was nontoxic to cells. [ Conclusion] The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein. In addition, the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells. The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.

关 键 词：新城疫 F基因 杆状病毒 鸡胚原代细胞 WPRE调控元件 

分 类 号：S852.65[农业科学—基础兽医学]