利用反向遗传学技术研究脊髓灰质炎病毒/柯萨奇病毒嵌合基因组的生物学特性  

Research on the biological character of poliovirus/coxsackievirus chimeric genome by reverse genetics

作　　者：李卫中[1] 洪敏[1] 吴文娟[1] 梁燕[1] 张雪梅[1] 寸韡[1] 马绍辉[1] 赵红玲[1] 李琦涵[1] 

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《云南大学学报（自然科学版）》2007年第S3期349-354,359,共7页Journal of Yunnan University(Natural Sciences Edition)

摘　　要：为探讨B_3型柯萨奇病毒(CVB_3)P_2基因区能否在功能上取代Ⅰ型脊髓灰质炎病毒(PV_1)的P_2基因区,构建包含PV_1/CVB_3嵌合体cDNA的真核表达质粒,其中PV_1的P_2基因区被来自于CVB_3的P_2基因区所代替.质粒转染真核细胞KMB-17后,导致了细胞病变效应,电镜观察到转染细胞的超微结构发生了改变并形成了病毒样颗粒(VLP).免疫电镜进一步证实了这些颗粒的存在.RT-PCR及中和实验结果表明质粒携带的外源基因发生转录并合成了病毒蛋白.蔗糖密度梯度离心和液闪仪计数实验显示转染细胞内标记蛋白的动态变化过程与脊髓灰质炎病毒疫苗株在正常细胞内的复制和装配过程类似.上述工作为进一步深入研究肠道病毒非结构蛋白在重组病毒的形成中所发挥的生物学作用奠定了基础.A chimeric plasmid(pSCS) was constructed , which includes a vector backbone from pcDNA_3 and a cDNA copy of Poliovirus with a substitution of P_2 gene region by the counterpart of Coxsackievirus. KMB- 17 cells changed their morphology after they were tranfected the chimeric plasmid through lipofec-tamine. Electronmicroscopy (EM) showed that there was a remarkable difference in ultrastructure between the transfected cells and the normal cells. Furthermore, some virus- like particles(VLP) were observed in these cells. Immunoelectronmicroscopy (IEM) furtherly proved the existence of these particles. Result of RT - PCR and neutrilization test showed that chimeric gene trancripted and translated into protein. Data from the sucrose density gradient centifugation and liquid scintillation counting test suggested that the dynamic alternations of labeled viral proteins in pSCS plasmid - transfected cells were similar to the replicative and assembling process of poliovirus in control cells. The above studying will contribute to the futher research of gene recombination between different virus.

关 键 词：反向遗传 脊髓灰质炎病毒 柯萨奇病毒 嵌合基因 病毒样颗粒 

分 类 号：S852.65[农业科学—基础兽医学]