表达传染性喉气管炎病毒gB主要抗原表位区域重组新城疫病毒的构建  

Constrution of a recombinant Newcastle disease virus expressing major antigen epitopes in gB of infectious laryngotracheitis virus

作　　者：孙娜娜[1] 王琪[1] 李慧昕[1] 刘胜旺[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽呼吸道病创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第6期414-417,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家蛋鸡体系(CARS-41-K12);公益性行业专项(201303033)

摘　　要：为构建以新城疫病毒(NDV)为活病毒载体表达传染性喉气管炎病毒(ILTV)g B主要抗原表位区域的重组病毒,本研究以ILTV LJS09株的DNA为模板,通过PCR技术扩增1 323 bp ILTV编码g B主要抗原表位的基因片段(1 bp^440 bp),将其插入到NDV感染性克隆p BR-FL中,构建含有ILTV g B主要抗原基因的NDV c DNA克隆p BR-FL-g B1-440。利用磷酸钙转染法,在辅助质粒p CI-neo-NP、p CI-neo-P和p CI-neo-L的共同作用下,将重组质粒转染于表达T7聚合酶重组痘病毒预感染的BSR-T7/5细胞,获得重组病毒r L-g B1-440。采用RT-PCR检测接种重组病毒的鸡胚尿囊液,结果表明重组病毒中含有相应的外源基因。利用g B的抗血清检测重组病毒中g B的表达,间接免疫荧光试验表明,在感染重组病毒的BSR-T7/5细胞中目的蛋白得到表达。本研究为研制和开发传染性喉气管炎重组新城疫二联活疫苗奠定基础。To construct the recombinant Newcastle disease virus （NDV） expressing the major antigen epitopes in gB of infectious laryngotracheitis virus （ILTV）, a 1,323 bp fragment encoding a truncated aal-aa440 of gB of ILTV LJS09 strain was amplified by PCR with specific primers and cloned into NDV infectious clone of pBR-FL to construct the pBR-FL-gB1-440. Together with helper plasmids, the recombinant plasmid pBR-FL-gB1-440 was transfected into the BSR-T7/5 cells which had been pre-infected with recombinant fowl poxvirus expressing T7 polymerase. Then, the recombinant virus （rL-gB1-440） was rescued, and the insertion of the gB gene fragment in rL-gB1-140 was verified by RT-PCR. Moreover, the expression of the truncated gB of ILTV in the rL-gB1-440 infected BSR-T7/5 cells was confirmed by indirect immunofiuorescence assay. The construction of rL-gB1-440 would facilitate the immuno-protection tests for development of the bivalent recombinant virus vaccine against NDV and ILTV infections in chickens.

关 键 词：重组新城疫病毒 传染性喉气管炎病毒 gB蛋白 主要抗原表位基因 

分 类 号：S852.65[农业科学—基础兽医学]