狂犬病病毒G蛋白昆虫细胞表达及其生物信息学分析  

Baculovirus Expression and Bioinformatic Analysis of Rabies Virus Glycoprotein

作　　者：王园园[1] 董晓琳[2] 孙钰[2] 李志萍[2] 靳朝[1] 夏志平[2] 

机构地区：[1]吉林大学动物医学学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130122

出　　处：《吉林农业大学学报》2016年第2期228-234,共7页Journal of Jilin Agricultural University

基　　金：国家科技基础条件平台建设计划项目(2011ZXJ09201-031);公益性行业(农业)科研专项(201103032)

摘　　要：利用RT-PCR方法从SRV9株细胞毒基因组RNA扩增G蛋白基因,然后将其克隆到p MD18-T载体上,将得到的重组克隆质粒SRV9G-p MD18T进行PCR和测序鉴定。结果表明:克隆的基因全长为1 609 bp,阅读框正确;将此SRV9株G基因亚克隆到供体质粒p Fast Bac Dual后获得了重组供体质粒SRV9G-p Fast Bac Dual;进而将重组供体质粒转化DH10Bac感受态细胞,经2次抗性、蓝白斑筛选及PCR分析后,获得重组穿梭质粒SRV9G-Bacmid;将其转染Sf9细胞48 h后,通过细胞形态学和重组杆状病毒负染电镜观察显示,杆状病毒转染成功;通过Western blot检测显示,Sf9细胞成功表达了SRV9 G蛋白。随后,利用Ex PASy服务器上的在线分析工具和生物信息学分析软件DNAStar对G蛋白的基本性质进行了分析。试验采用昆虫细胞Bac-toBac杆状病毒表达系统,成功表达出G蛋白,并对其进行了生物信息学分析,为蛋白纯化及狂犬病诊断试剂盒的进一步研制奠定了基础。In this study, glycoprotein gene of SRV9 cellular strain amplified with RT-PCR was cloned into pMD18-T vector, and the recombinant cloning plasmid was identified by PCR and se- quencing. The results showed that the constructed recombinant plasmid contained the sequence of SRV9 glycoprotein gene, which has a correct reading frame and reaches a length of 1 609 bp. The baculovirus transfer vector of pFastBacDual containing the gene of interest was constructed and transformed into DHIOBac which contains a shuttle vector of bacmid, and the colonies were identified by resistance screening, blue-white selection and PCR analysis, which successfully constructed recom- binant shuttle plasmid SRV9G-Bacmid; after Sf9 cells were transfected by recombinant shuttle plasmid for 48 h, the observation of cell morphology and negative-stain electron microscopy showed efficient gene transfer; Western blot experiments proved that the recombinant haculovirus containing SRV9G gene could express G proteins in insect cells. Basic properties of the glycoprotein were analyzed on the ExPASy server and DNAStar. This study successfully expressed SRV9 G proteins and obtained the basic properties of SRV9 glycoprotein, which lays foundation for the purification of the protein and further development of Rabies diagnosis kit.

关 键 词：狂犬病病毒G蛋白 杆状病毒表达系统 生物信息学分析 SRV9株 

分 类 号：S852.65[农业科学—基础兽医学]