以杆状病毒为载体在鸡原代骨骼肌细胞中表达IBDV VP2基因

The expression of IBDV VP2 in chicken primary myoblast cells using the baculovirus vector

摘要:

摘要：【目的】本研究旨在构建在鸡原代骨骼肌细胞中表达IBDV病毒VP2基因的重组杆状病毒。【方法】从IBDV适应细胞毒中提取RNA，用RT-PCR技术扩增VP2基因，将其克隆到自主构建的杆状病毒转移载体的CMV启动子之下，通过Bac-to-Bac系统获得VP2重组Bacmid，并将其转染Sf9昆虫 细胞，获得了VP2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72h后裂解细胞收获蛋白。【结果】蛋白样品经SDS-PAGE和Western blot证实VP2蛋白获得表达，分子量约48kDa，与预测蛋白大小一致，且能被IBDV阳性血清所识别。【结论】重组杆状病毒可以有效地将VP2基因导入鸡原代细胞，并在CMV的启动下表达具有抗原性的VP2蛋白，本研究为研制IBDV及其他重要禽类传染病的杆状病毒载体疫苗奠定了基础。

Abstract:

Abstract: [Objective] To construct the recombinant baculovirus expressing Infectious bursal disease(IBDV) VP2 gene in the chicken primary myoblast cells.[Methods] A proteinase K digestion and phenol-chloroform extraction method was used to extract dsRNA genome from IBDV. VP2 gene was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) with the genome RNA as template. The pFastBac-pCMV-VP2 baculovirus transfer vector was constructed by inserting VP2 gene under the immediate-early promoter of cytomegalovirus. The VP2 recombinant bacmid was obtained by Bac-to-Bac system and transfected sf9 insect cell to acquire VP2 recombinant baculovirus. After amplification of recombinant baculovirus on cell passages, the recombinant virus was seeded on chicken primary myoblast cells with 50 multiplicity of infection (MOI), and the cells were harvested at 72 hours after infection. [Results]Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) and Western blot results showed that the VP2 gene was successfully expressed in chicken primary myoblast cells. The product was a 48kDa protein and could be recognized by anti-IBDV serum.[Conclusion] The recombinant baculovirus could efficiently delivery IBDV VP2 gene into chicken primary cells and that CMV, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct the expression of VP2 antigen protein.The research can be a potential basis for the development of baculovirus vector vaccines for IBDV and other avian infectious disease.