摘要
为开发猪O型口蹄疫病毒(FMDV)病毒样颗粒(VLPs)基因工程亚单位疫苗,试验参考GenBank中登录的FMDV毒株基因序列(登录号:JN998085),设计针对VP 1、VP 2、VP 3和VP 44个基因片段的特异性引物,以O型FMDV O/MYA98/XJ/2010毒株的cDNA序列为模板,对目的基因进行PCR扩增;将获得的VP 3、VP 1和VP 4、VP 2基因片段分别插入2个杆状病毒供体质粒(pFastBacDual)的p10和pH双元启动子中,构建pFBD-VP3-VP1和pFBD-VP4-VP22个重组转座质粒;将验证正确的2个重组转座质粒分别转化含有穿梭载体(Bacmid)的大肠杆菌DH10Bac感受态细胞,获得2个重组杆粒rBacmid-VP3-VP1和rBacmid-VP4-VP2,经验证正确后,对其进行扩增和提取,将其分别转染Sf9贴壁昆虫细胞,构建2个重组杆状病毒rvAc-VP3-VP1和rvAc-VP4-VP2;2个重组杆状病毒共同感染悬浮培养的Sf9昆虫细胞,利用杆状病毒表达系统在昆虫细胞内对4个基因进行表达,目的蛋白通过间接免疫荧光试验(IFA)、SDS-PAGE、Western blotting及透射电镜(EM)进行检测。结果显示,本研究成功构建2株分别表达FMDV VP1、VP2、VP3和VP44个结构蛋白的重组杆状病毒;特异性抗体检测发现,4个蛋白VP1~VP4均成功表达,且具有良好的特异性反应;4个蛋白在Sf9昆虫细胞内能够完成自我组装,形成与天然病毒结构相似的VLPs,直径大小在25~30 nm。本研究利用共感染表达方式在Sf9昆虫细胞内成功制备出FMDV病毒颗粒,为开发高效安全的FMDV基因工程亚单位疫苗开辟了一条新思路。
To develop the virus-like particles(VLPs)genetic engineering subunit vaccine for swine O-type foot-and-mouth disease virus(FMDV),according to the FMDV complete gene sequence available in GenBank(accession No.:JN998085),four pairs of specific primers were designed to amplify VP1,VP2,VP 3 and VP 4 genes of FMDV O/MYA98/XJ/2010 strain by PCR.VP3,VP 1 and VP4,VP 2 gene were cloned into p10 and pH promoters of pFastBac Dual vector,respectively.The recombinant plasmid pFBD-VP3-VP1 and pFBD-VP4-VP2 were obtained and identified.Two recombinant bacmid of rBacmid-VP3-VP1 and rBacmid-VP4-VP2 were generated after the E.coli DH10Bac competent cell was transformed by the two recombinant plasmid,respectively.Two recombinant baculovirus of rvAc-VP3-VP1 and rvAc-VP4-VP2 were successfully rescued by transfected two recombinant bacmids into Sf9 insect cells.Two recombinant baculoviruses were coinfected with suspension cultured Sf9 insect cells,and VP1,VP2,VP 3 and VP 4 genes were expressed via Bac-to-Bac expression system in Sf9 insect cells.The target proteins were identified and analyzed with specific positive antibody through IFA,SDS-PAGE and Western blotting,and the target proteins were checked as VLPs via by electron microscopy(EM).The results showed that two recombinant baculoviruses expressing four structural proteins of FMDV VP1,VP2,VP3 and VP4 were successfully constructed.The specific antibodies detection results showed that four proteins VP1,VP2,VP3 and VP4 were successfully expressed with good specificity.Four proteins could self-assemble in Sf9 insect cells and form VLPs with a diameter of 25-30 nm similar to that of natural viruses.In this study,FMDV VLPs were successfully prepared in Sf9 insect cells by coinfection expression,which laid a foundation for further developing efficient and safe FMDV gene engineering subunit vaccine.
作者
刘汉平
LIU Hanping(Shanghai Research Center of Veterinary Biological Engineering and Technology,Shanghai HILE Bio-Technique Co.,Ltd.,Shanghai 201403,China)
出处
《中国畜牧兽医》
CAS
北大核心
2019年第11期3350-3357,共8页
China Animal Husbandry & Veterinary Medicine
基金
上海兽用生物制品工程技术研究中心(19DZ2284700)
