ASFV抗原重组腺病毒的构建及免疫保护效果  

Construction and immunoprotective effect of recombinant adenovirus containing ASFV antigen

作　　者：惠丽丽 岳慧贤 蒋依倩 张艳艳[1] 齐宇 李思齐 付林 李其炫 扈荣良 HUI Lili;YUE Huixian;JIANG Yiqian;ZHANG Yanyan;QI Yu;LI Siqi;FU Lin;LI Qixuan;HU Rongliang(Changchun Veterinary Research Institute,Chinese Academy Agricultural Sciences,Changchun 130122,China)

机构地区：[1]中国农业科学院长春兽医研究所,长春130122

出　　处：《黑龙江畜牧兽医》2024年第21期100-106,128,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目(2021YFD1801400)。

摘　　要：为了构建非洲猪瘟病毒(African swine fever virus,ASFV)抗原重组腺病毒并探讨不同抗原的免疫原性,试验以ASFV基因Ⅱ型SY18株基因组为模板,采用无缝连接技术构建13种带有His标签的ASFV抗原重组腺病毒,通过间接免疫荧光技术鉴定重组腺病毒在HEK-239AD细胞中的表达情况,并用构建的13种重组腺病毒与前期构建的25种重组腺病毒分别于试验第0,14,28天免疫小鼠,200μL/只(重组腺病毒稀释至1×10^(8)TCID_(50)/mL)。第3次免疫后第7天,通过摘眼球方法采集血液,制备血清,利用血清中和试验筛选出5种效果较好的可刺激动物机体产生ASFV抑制性抗体的抗原,并将这5种重组腺病毒混合后免疫猪只,每种重组腺病毒(稀释至1×10^(8)TCID_(50)/mL)用量为0.5 mL/头;首免(第0天)后第14天进行二免,二免后第14天口服ASFV基因Ⅱ型SY18株(效价为1×10^(4)TCID_(50)/mL)200μL攻毒,探究重组腺病毒免疫保护效果。结果表明:本次试验构建的13种重组腺病毒均能在HEK-239AD细胞中稳定表达;从免疫小鼠血清中和试验筛选出5种抑制ASFV效果较好的重组腺病毒为rAdCMV-D129L、rAdCMV-EP84R、rAdCMV-E146L、rAdCMV-NP419L、rAdCMV-S273R。5头免疫重组腺病毒的猪均在攻毒后延迟发病,其中4头猪最终死亡,1头存活,保护率为20%,免疫PBS的猪只在攻毒后发病快,并全部死亡。说明ASFV抗原重组腺病毒可以刺激机体产生免疫反应,但用经过血清中和试验筛选出来的5种抗原制备的重组腺病毒无法提供完全保护。In order to construct African swine fever virus(ASFV)antigen-containing recombinant adenovirus and to explore the immunogenicity of different antigens,in the experiment,13 recombinant adenoviruses with His-tagged ASFV antigens were constructed by seamless ligation using the genome of ASFV genotype II strain SY18 as a template;the expression of the recombinant adenoviruses in HEK-239AD cells was characterized by indirect immunofluorescence.Mice were immunized with 13 recombinant adenoviruses constructed in this study and 25 recombinant adenoviruses constructed in the previous period on days 0,14,and 28 of the experiment,respectively with the injection volume of 200μL/head(recombinant adenoviruses were diluted to 1×10^(8)TCID_(50)/mL),respectively.On day 7 after the third immunization,blood was collected by eyeball removal and serum was prepared.The serum neutralization test was used to screen the top five antigens that could stimulate the production of inhibitory antibodies to ASFV,and five recombinant adenoviruses that could produce inhibitory antibodies were combined to immunize pigs on days 0 and 14 of the test,respectively.The dosage of each recombinant adenovirus was 0.5 mL/head(recombinant adenovirus was diluted to 1×10^(8)TCID_(50)/mL);the second immunization was constructed on the 14th day after the first immunization(0 day);on the 14th day after the second immunization,200μL of ASFV gene type II strain SY18(potency 1×10^(4)TCID_(50)/mL)was orally administered for virus challenge;the immunoprotective effect of the recombinant adenoviruses was investigated.The results showed that all 13 recombinant adenoviruses constructed in the experiment were stably expressed in HEK-239AD cells;five recombinant adenoviruses rAdCMV-D129L,rAdCMV-EP84R,rAdCMV-E146L,rAdCMV-NP419L,and rAdCMV-S273R inhibited ASFV more effectively were screened out from the sera of immunized mice;after challenge,all five pigs immunized with the recombinant adenovirus had delayed onset of disease;four of them eventually died and one survive

关 键 词：非洲猪瘟病毒 重组腺病毒 抗原鉴定 病毒复制 抑制性抗体 

分 类 号：S852.65[农业科学—基础兽医学]