表达鸡传染性喉气管炎病毒gB基因的重组鸡痘病毒rFPV-ILTVgB的理化特性及遗传稳定性研究  

The physical and chemical properties and genetic stability of recombinant fowlpox virus expressing gB gene of avian infectious laryngotracheitis virus

作　　者：李诗 他蕾 许霄 秦涛[1,2] 陈素娟 彭大新[1,2] LI Shi;TA Lei;XU Xiao;QIN Tao;CHEN Su-juan;PENG Da-xin(College of Veterinary Medicine Yangzhou University,Yangzhou 225000,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225000 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225000

出　　处：《中国预防兽医学报》2021年第8期806-811,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：十三五重点研发计划(2017YFD0500701)。

摘　　要：为检测各理化因子对表达鸡传染性喉气管炎病毒(ILTV)gB基因的重组鸡痘病毒rFPV-ILTVgB的影响,并探究其在体内体外的遗传稳定性,本研究将rFPV-ILTVgB经热、酸、碱、氯仿、乙醚和胰酶分别处理后,接种于鸡胚成纤维细胞(CEF)中,测定病毒的半数感染量(TCID50),结果显示:rFPV-ILTVgB经55℃水浴30 min或60℃水浴15 min可被完全灭活;经pH3~pH9的酸碱作用或经24 h的乙醚作用,病毒滴度变化不明显;经胰蛋白酶或氯仿处理后,病毒滴度下降明显;将rFPV-ILTVgB在CEF中连续培养30代,取第0、5、10、15、20、25、30代rFPV-ILTVgB分别扩增g B基因并测序,通过间接免疫荧光试验鉴定gB蛋白的表达,结果显示:rFPV-ILTVgB能在CEF细胞中稳定传代,gB基因序列在传代过程中未发生任何突变,gB蛋白稳定表达;同时将rFPV-ILTVgB在SPF鸡体内连续传代,结果显示,rFPV-ILTVgB在SPF鸡体内的传代过程中,gB基因的阳性检出率逐渐降低,对鸡体的致病性逐渐降低,不存在毒力返强的可能,符合生物制品的相关要求。本研究选取表达ILTV优势流行株gB基因的rFPV-ILTVgB,首次从分子生物学和遗传学等角度研究该疫苗候选株,为后续该疫苗的生产、运输提供参考。To detect the effects of various physical and chemical factors on recombinant fowlpox virus rFPV-ILTVgB expressing gB gene of infectious laryngotracheitis virus, and to explore its genetic stability in vivo and in vitro, rFPV-ILTVgB was inoculated into chicken embryo fibroblasts(CEF) after being treated with heat, acid, alkali, chloroform, ether and trypsin, and the50% tissue culture infective dose(TCID50) was determined. The results showed that rFPV-ILTVgB could be completely inactivated by 55℃ water bath for 30 min or 60℃ water bath for 15 min. The virus titer did not change significantly after being treated with acid-base of p H3-p H9 or with ether for 24 hours, while the virus titer decreased significantly after trypsin or chloroform treatment.The r FPV-ILTVg B virus was continuously cultured on CEF for 30 passages. The 0, 5, 10, 15, 20, 25, 30 passages of rFPV-ILTVg B were amplified and sequenced, and the expression of g B protein was identified by indirect immunofluorescence assay. The results showed that rFPV-ILTVg B could be stably passaged in CEF cells, there was no mutation in g B gene sequence, and gB protein was stably expressed. Meanwhile, rFPV-ILTVg B was continuously passaged in SPF chickens. The results showed that the positive detection rate of gB gene decreased gradually, and the pathogenicity to chickens decreased gradually during the passage of r FPVILTVg B in SPF chickens, and there was no possibility of virulence reversion, which met the relevant requirements of biological products. In this study, r FPV-ILTVg B expressing the g B gene of the dominant epidemic strain of ILTV was selected as the vaccine candidate strain and studied from the point of view of molecular biology and genetics for the first time, which provides reference for subsequent production and transportation.

关 键 词：传染性喉气管炎 重组鸡痘病毒 GB基因 理化特性 遗传稳定性 

分 类 号：S855.3[农业科学—临床兽医学]