甲型肝炎病毒病毒样颗粒的制备  

Preparation of hepatitis A virus-like particles

作　　者：沈智俊[1,2] 吴杰[1] 解庭波[1] 吕宏亮[1] 王云[3] 陈新文[3] 严家新[1] 沈智俊 王云[1] 陈新文[1] 

机构地区：[1]武汉生物制品研究所有限责任公司基因工程室,湖北武汉430060 [2]湖北省中山医院细胞诊疗研究室,湖北武汉430033 [3]中国科学院武汉病毒研究所肝炎病毒室,湖北武汉430071

出　　处：《中国生物制品学杂志》2013年第4期441-447,共7页Chinese Journal of Biologicals

摘　　要：目的制备甲型肝炎病毒（Hepatitis A virus,HAV）病毒样颗粒（Virus-like particles,VLPs）,为研制HAV VLPs疫苗奠定基础。方法利用RT-PCR法从HAV HM175-clone4株中扩增P1-2A、P2和P3基因片段。将P1-2A和P3基因亚克隆至pFastBacDual载体,构建重组表达质粒pFastBacDual-P1-P3,转化大肠杆菌DH10BacTM,构建重组杆状病毒穿梭质粒Bacmid-P1-P3,转染昆虫细胞sf-9进行重组杆状病毒的包装。重组杆状病毒vAcP1P3扩增后,感染昆虫细胞Tn-5,培养72 h后,收获表达产物,经原位免疫荧光、EIA、Western blot法及透射电镜检测HAV VLPs的表达。表达产物经超滤及蔗糖密度梯度离心纯化。结果重组表达质粒和重组穿梭质粒分别经双酶切和PCR鉴定证明构建正确。第2、3、4代重组杆状病毒的滴度分别为1.58×105、2.13×107、3.98×107TCID50/ml。vAcP1P3转染sf-9细胞72 h后,可见HAV蛋白的表达;vAcP1P3转染Tn-5细胞后,表达的HAV抗原主要存在于沉淀中,且含量明显高于野生型杆状病毒沉淀样品（P〈0.001）;Wentern blot结果表明,与野生型杆状病毒组相比,HAV VLPs样品中VP3的含量相对较少,HAV VLPs样品可见相对分子质量约30 700的VP1片段及相对分子质量约27 700的VP3片段;透射电镜观察可见大小约50 nm的VLPs颗粒,略大于天然HAV颗粒。纯化的HAV蛋白主要存在于40%~50%的蔗糖梯度中,提示有VLPs形成。结论成功制备了HAV VLPs,为甲肝新型疫苗的研制奠定了基础。Objective To prepare the virus-like particles（VLPs） of hepatitis A virus（HAV）,and lay a foundation of development of HAV VLPs as vaccine.Methods P1-2A,P2 and P3 genes were amplified from HAV HM175-clone4 by RT-PCR.P1-2A and P3 genes were subcloned into vector FastBacDual,and the constructed recombinant plasmid pFastBacDual-P1-P3 was transformed to E.coli DH10BacTM.The obtained recombinant shuttle plasmid Bacmid-P1-P3 was transfected to sf-9 cells for packaging of recombinant baculovirus.The recombinant baculovirus vAcP1P3 after proliferation was infected to insect cells Tn-5 and cultured for 72 h.The expressed HAV VLPs were determined by in situ IFA,EIA,Western blot and transmission electron microscopy,and purified by ultrafiltration and sucrose density gradient centrifugation.Results Recombinant expression vector and recombinant shuttle plasmid were constructed correctly as proved by restriction analysis and sequencing.The titers of recombinant baculovirus of passages 2,3 and 4 were 1.58 × 105,2.13 × 107 and 3.98 × 107 TCID50 / ml respectively.The expression of HAV protein was observed in sf-9 cells 72 h after transfection with vAcP1P3.The expressed HAV antigen in Tn-5 cells transfected with vAcP1P3 mainly existed in precipitate,of which the content was significantly higher than that in wild HAV（P 0.001）.Western blot showed that the VP3 content in HAV VLPs was lower than that in wild baculovirus.VP1 and VP3 fragments,with relative molecular masses of about 30 700 and about 27 700 respectively,were observed in HAV VLPs.VLPs at a mean diameter of about 50 nm were observed by transmission electron microscopy,which were slightly larger than natural HAV particles.Purified HAV protein mainly existed in 40% ~ 50% sucrose density gradient,indicating formation of VLPs.Conclusion HAV VLPs were prepared successfully,which laid a foundation of development of novel hepatitis A vaccine.

关 键 词：甲型肝炎病毒 病毒样颗粒 杆状病毒表达系统 

分 类 号：Q55[生物学—生物化学]