摘要
旨在比较火鸡疱疹病毒(HVT)亚克隆对构建活载体疫苗的影响。本研究采用同源重组方法,在HVT Fc126的UL55与UL56基因间的非编码区插入MiniF序列,构建重组病毒;采用细菌人工染色体(BAC)技术,分别提取病毒DNA后转入DH10B感受态细胞中,构建3个对应的BAC;将传染性法氏囊病毒(IBDV)的VP2表达盒分别与3株BAC的DNA共转染鸡胚成纤维细胞(CEF),进行重组病毒的拯救;将重组活载体、亲本毒株及商品化重组活载体的生长动力学进行比较;将3株重组活载体接种SPF鸡后,在免疫后第3、4、5、6周等共4个时间点采集血清进行琼脂扩散试验。结果:获得3个纯化的HVT-MiniF亚克隆病毒,然后获得3株对应的HVT BAC,分别命名为HVT BAC-C1、HVT BAC-C2和HVT BAC-C3。限制性片段长度多态性(RFLP)鉴定发现这3个BAC亚克隆的基因组主体序列几乎一致,略有差异;进一步获得3株相对应的VP2重组HVT活载体,分别命名为HVT-VP2-C1、HVT-VP2-C2、HVT-VP2-C3;生长动力学结果表明,3株VP2重组病毒与2株对照病毒的滴度在6、24、36和72 h存在显著性差异(P<0.05),在12和48 h无明显差异(P>0.05);琼脂扩散试验结果发现3个亚克隆的重组病毒均能产生较高抗体,但都没有达到对照商品疫苗的水平;3个亚克隆重组病毒之间产生的抗体存在明显差异,其中HVT-VP2-C2免疫鸡后产生的抗体最高。综上,HVT在传代过程中可能易出现基因变异,提示在构建重组活载体疫苗时,应充分考虑不同亚克隆活载体的免疫效力差异,做好种毒纯化。
This study was to compare the influence of Herpesvirus of Turkey(HVT)sub-clones on the generation of live vector vaccines.Homologous recombination was to insert the MiniF into the non-coding region between UL55 and UL56 of HVT strain Fc126 to generate HVT-MiniF recombinants.After three rounds of plaque picking,three recombinant HVT sub-clones were obtained.Then,using the bacterial artificial chromosome technology,the viral DNA of MiniF recombinant HVT was extracted and was transferred into DH10B competent cells to construct three HVT bacterial artificial chromosomes(BAC).After co-transfection of the expression cassette harboring the infectious bursal disease virus(IBDV)VP2 and the BAC DNA into CEFs(Chicken Embryo Fibroblasts,CEF),the growth kinetics of the vectored HVT re⁃combinants,the parental strain and the commercial vaccine strain were tested.Finally,serum samples were collected from specific pathogen free(SPF)chickens at 3,4,5 and 6 weeks after immunization with the three recombinant live vectors so as to detect the antibody against IBDV with the method of agar gel precipitation test(AGP).The result showed that we first obtained three purified HVT-MiniF subcloned vi⁃ruses,followed by three corresponding HVT bacterial artificial chromosomes(BACs)which were termed as HVT BAC-C1,HVT BAC-C2,and HVT BAC-C3,respectively.The RFLP(Restriction Fragment Length Polymorphism,RFLP)assay showed the same major fragments,but with differences between these three BACs.We obtained three recombinants HVT live vectors,namely HVT-VP2-C1,HVT-VP2-C2,and HVT-VP2-C3.The results of the growth kinetics showed that there were significant differences between these strains at 6,24,36 and 72 h(P<0.05),but no significant differences at 12 and 48 h(P>0.05).All three HVT recombinants from the three sub-clones induced high AGP antibodies which was still lower than the level of the commercial HVT vectored vaccines.There were obvious differences in AGP antibodies induced by the three recombinants,in which HVT-VP2-C2 produced the highest level of antibody.In summary,this study indi⁃cated that mutations might be likely to happen in the HVT genome during serial passages,highlighting the importance of selection of vaccine strains with consideration of the effect of sub-clone variations on the efficacy of HVT live-vectored vaccines.
作者
谢丽华
王志胜
童玲
栗环环
郑亚婷
刘娅梅
张传健
郭容利
Armando Mario DAMIANI
呙荣兵
黄腾
王继春
XIE Lihua;WANG Zhisheng;TONG Ling;LI Huanhuan;ZHEN Yating;LIU Yamei;ZHANG Chuanjian;GUO Rongli;Armando Mario DAMIANI;GUO Rongbing;HUANG Teng;WANG Jichun(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China;Guotai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Science,Nanjing 210014,China;Faculty of Medical Sciences,National University of Cuyo,Mendoza 5500,Argentina;Longchi Street Husbandry and Veterinary Service Center of Luhe District,Nanjing 211505,China)
出处
《畜牧与兽医》
北大核心
2025年第6期92-97,共6页
Animal Husbandry & Veterinary Medicine
基金
广西科技重大专项(桂科AA23062050)。
关键词
火鸡疱疹病毒
亚克隆
活载体疫苗
细菌人工染色体
免疫效力
turkey herpesvirus
sub-clone
live vector vaccine
bacterial artificial chromosome
immunization efficacy
