摘要
为构建以新城疫病毒(NDV)为活毒载体表达鸡传染性支气管炎病毒(IBV)S基因的重组病毒,本研究利用RT-PCR技术,以IBV的Massachusetts41株RNA为模板,通过RT-PCR扩增得到IBV S基因(3534bp),将其插入到NDV感染性克隆pBRN-FL中,构建了含有IBV S基因的重组NDV cDNA克隆pBRN-FL-IBVS。利用磷酸钙转染法,在辅助质粒pBS-NP、pBS-P和pBS-L的共同作用下,将pBRN-FL-IBVS转染表达T7聚合酶重组痘病毒感染的BSR细胞,救获重组NDV(rL-IBVS)。采用RT-PCR检测接种重组病毒的鸡胚尿囊液,结果表明rL-IBVS中含有相应外源基因。IFA试验表明,rL-IBVS可与鸡抗IBV的高免血清发生特异性反应,证明S蛋白在感染的BSR细胞中得到表达。其鸡胚平均致死时间、脑内致病指数和静脉内致病指数等指标显示rL-IBVS保持了亲本疫苗株高滴度的鸡胚生长特性和低致病力特性。本研究采用反向遗传操作技术构建了表达IBV S蛋白的重组NDV,为进一步研制IBV和NDV的重组基因工程活载体疫苗奠定了基础。
The S gene（3 534 bp） encoding the spike protein of infectious bronchitis virus（IBV） Massachusetts 41 strain was amplified by RT-PCR with specific primers and cloned into Newcastle disease virus（NDV） infectious clone of pBRN-FL to construct pBRN-FL-IBVS.The recombinant plasmid was transfected,together with helper plasmid,into the BSR cells that had been pre-infected by recombinant fowl poxvirus expressing T7 polymerase.Recombinant virus rL-IBVS was rescued and insertion of S gene in rL-IBVS was verified by RT-PCR.Expression of S protein in BSR cells infected with rL-IBVS was confirmed by indirect immunofluoresence（IFA）.The results of MDT,ICPI and IVPI demonstrated rL-IBVS replicated to a titer similar to that of parental NDV LaSota vaccine strain in chicken embryos and retained low pathogenicity of LaSota strain.The recombinant virus constructed by applying reverse genetic techniques could be further developed as a novel live viral vector vaccine against both infectious bronchitis and Newcastle disease.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2010年第5期334-337,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家青年科学基金(30800816)
关键词
重组新城疫病毒
传染性支气管炎病毒
S基因
recombinant Newcastle disease virus
infection bronchitis virus
S gene
