融合表达TM-1基因和gD基因重组腺病毒的构建及其免疫效果初步评价  

Construction and evaluation of recombinant adenovirus fusion expressing the TM-1 gene and the gD gene

作　　者：李小慧 高珂珂 张东超 弓建芳 林静 李昕 孟佳丽 尚翠玲 金天明 LI Xiao-hui;GAO Ke-ke;ZHANG Dong-chao;GONG Jian-fang;LIN Jing;LI Xin;MENG Jia-li;SHANG Cui-ling;JIN Tian-ming(College of Animal Science and Veterinary Medicine,Tianjin Agriculture University,Tianjin 300384,China)

机构地区：[1]天津农学院动物科学与动物医学学院

出　　处：《中国兽医学报》2018年第8期1473-1478,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31572492,31072109);天津市优秀科技特派员资助项目(16JCTPJC45000);天津市优秀科技特派员资助项目(2016078);天津市自然科学基金重点资助项目(12JCZDJC22100);天津市农委农业科技示范推广资助项目(201202110);天津市兽医生物技术优秀科研创新团队资助项目(TD12-5019);天津市兽医学重点学科“领军人才培育计划”人选资助项目

摘　　要：为研究鸡毒支原体病和鸡传染性喉气管炎2种禽类接触性呼吸道传染病二联基因工程活载体疫苗，试验中选取鸡毒支原体TM-1基因和鸡传染性喉气管炎gD基N，经鉴定获得阳性pMD-TM-1和pMD-gD克隆质粒，酶切胶回收目的基因后分别克隆至穿梭载体构建重组pDC315-TM-1、pDC315-g D和pDC315-TM1-gD。将构建成功的重组穿梭载体和骨架质粒同时转染293细胞，经荧光显微观察、RT-PCR和Western blot检测，成功包装出能表达TM-1蛋白、gD蛋白和TM-1-gD融合蛋白的重组腺病毒pBH-TM-1、pBH-gD和pBH-TM-1-gD。Vivapure AdenoPACKTM法纯化病毒粒子，以TCID50法确定pBH-EGFP、pBH-TM1、pBH-gD和pBH-TM-1-gD的滴度分别为10T10 TCID50／mL、10^10.125 TCID50／mL、10^9.75 TCID50／mL、10^10.25 TCID50／mL，符合后续动物试验所需滴度要求。动物试验肌内免疫SFP雏鸡结果显示：重组腺病毒pBH-TM-1-gD组与常规疫苗组免疫效果无显著性差异（P〉0．05），可以成为一种替代抗生素的生物治疗方式。Abstract,To provide an experimental reference for the further research on the bivalent genetic vector vaccine of CRD and ILT, TM-1 gene and gD gene were selected to construct a recombinant ad- enovirus fusing expressing the TM-I-gD protein. After the cloning vector pMD-TM-1 and pMD- TM-1 identified by double digestion and sequencing analysis,T4 ligase was used to link the target genes into the shuttle vector to construct pDC315-TM-1、pDC315-gD and pDC315-TM1-gD, respec tively. Then these recombinant shuttle vectors contrusted successfully were co-transfected into 293 cells with constructed pBHGlox（delta）E1,3cre simultaneously in the help of lipofection and the recombinant adenoviruses were tested by fluorescence detection, RT-PCR and Western blot and purified by Vivapure AdenoPACKTM. TCIDs0 detected viruses titers. 10^10 TCID50/mL, 10^10.125TCID50/mL animal expe adenoviruse ,109.75 TCID50/mL riment. The results s had the ability 0 and 10^10.25 TCID50/mL, respectively. It was of indirect ELISA and spleen index showed f inducing immune response in a sufficient enough for that the re follow-up corn level compared withconventional vaccines. Above all, the recombinant adenovirus fusion expressing the TM-1 gene of MG and the gD gene of ILTV was constructed successfully,which had an satisfactory immunological efficacy it provided theoretical and experimental supports for the further study.

关 键 词：TM-1基因 GD基因 重组腺病毒 融合表达 免疫效果 

分 类 号：S852[农业科学—基础兽医学]