【作者】 张婷；

【导师】 苏帅；

【作者基本信息】 山东农业大学 ， 兽医（专业学位）， 2021， 硕士

【摘要】 禽腺病毒(Fowl adenovirus,FAdV)包括5个种(A-E种)、12个血清型(1-8a,8b-11)。FAdV有多种血清型在我国鸡群中流行。近年来,血清2型禽腺病毒(FAdV-2)、血清4型禽腺病毒(FAdV-4)、血清8b型禽腺病毒(FAdV-8b)在鸡群内流行趋势愈加严重,给疫病防控带来困难。纤突蛋白2(Fiber2)位于禽腺病毒表面,是禽腺病毒的重要结构蛋白,Fiber2含有与病毒的感染性有关的中和抗原表位,能够识别宿主的特异性受体。利用昆虫杆状病毒表达系统对FAdV-2-Fiber2、FAdV-4-Fiber2、FAdV-8b-Fiber2蛋白进行表达并进一步研究了重组蛋白的免疫原性。本研究根据Gen Bank上收录的FAdV-2、FAdV-4、FAdV-8b的基因序列,设计含有XholⅠ和K pnl酶切位点的上下游引物,扩增得到FAdV-2-Fiber2、FAdV-4-Fiber2、FAdV-8b-Fiber2编码序列。将其克隆到真核表达载体p Fast Bacdaul中,构建含有FAdV-2-Fiber2、FAdV-4-Fiber2、FAdV-8b-Fiber2的杆状病毒表达供体质粒。将其转化入DH10bac感受态细胞,筛选得到重组杆状病毒质粒。将重组质粒转染到昆虫细胞中获得重组杆状病毒。通过间接免疫荧光验证FAdV-2-Fiber2、FAdV-4-Fiber2、FAdV-8b-Fiber2重组蛋白在杆状病毒感染的昆虫细胞中表达,结果表明成功构建了能够稳定表达Fiber2蛋白的重组杆状病毒。利用超声破碎提取FAdV-2-Fiber2、FAdV-4-Fiber2、FAdV-8b-Fiber2重组蛋白,免疫SPF鸡,制备得到Fiber2重组蛋白的鸡单因子血清。用ELISA检测方法对SPF鸡抗血清检测,结果表明,免疫重组蛋白的鸡血清抗体效价分别为1:4096、1:16384、1:4096,说明获得的重组Fiber2蛋白有很好的免疫原性。病毒中和试验检测重组蛋白抗血清在LMH细胞上中和FAdV的活性,结果表明,三种重组蛋白抗血清的中和效价分别为:1:158、1:133、1:223,说明抗血清能中和FAdV对LMH细胞的感染。利用超声破碎提取FAdV-2-Fiber2、FAdV-4-Fiber2、FAdV-8b-Fiber2重组蛋白,免疫昆明白小鼠,制备得到Fiber2重组蛋白的鼠单因子血清。间接免疫荧光试验测得鼠抗血清在1:200倍稀释时,仍具有较好的活性且特异性良好。综上:本研究成功构建了能够稳定表达Fiber2蛋白的重组杆状病毒,进一步证实了表达的Fiber2蛋白具有良好的免疫原性。研究结果为FAdV的Fiber2蛋白生物学功能的研究奠定了物质基础,也为FAdV的免疫防控提供科学参考。更多还原

【Abstract】 Fowl adenovirus(FAdV)includes 5 species(A-E species)and 12 serotypes(1-8a,8b-11).Several serotypes of FAdV are prevalent in chickens in China.In recent years,the prevalence of serum type 2 avian adenovirus(FAdV-2),serum type 4 avian adenovirus(FAdV-4)and serum type 8b avian adenovirus(FAdV-8b)in chickens has become more and more serious,which brings difficulties to the prevention and control of the disease.Fiber2,located on the surface of avian adenovirus,is an important structural protein of avian adenovirus.Fiber2 contains neutralizing epitopes related to the infectivity of the virus and can recognize host specific receptors.FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 proteins were expressed by insect baculovirus expression system and the immunogenicity of the recombinant protein was further studied.According to this study included on Gen Bank FAdV-2,FAdV-4,FAdV-8b gene sequence,design contains Xhol Ⅰ and Kpn l enzyme loci of upstream and downstream primers,amplification FAdV-2-Fiber2,FAd-4-Fiber2,FAdV-8b-Fiber2 coding sequence.The recombinant plasmid was cloned into eukaryotic expression vector p Fast Bacdaul to construct baculovirus donor plasmid containing FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2.Recombinant baculovirus plasmid was obtained by transforming it into DH10 Bac competent cells.Recombinant baculovirus was obtained by transforming the recombinant plasmid into insect cells.The expression of recombinant FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 in insect cells infected with baculovirus was verified by indirect immunofluorescence,and the results showed that the recombinant baculovirus with stable expression of Fiber2 protein was successfully constructed.The recombinant proteins of FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 were extracted by ultrasonic crushing,and SPF chickens were immunized to prepare chicken polyclonal antibodies against recombinant Fiber2 protein.ELISA method was used to detect SPF chicken antiserum.The results showed that the titer of chicken serum antibody of the immune recombinant protein was 1:4096,1:16,384 and 1:4096,respectively,indicating that the obtained recombinant Fiber2 protein had good immunogenicity.The neutralization assay was used to detect the neutralization and FAdV activity of the recombinant protein antisera on LMH cells.The results showed that the neutralization titers of the three recombinant protein antisera were 1:158,1:133 and 1:2233,respectively,indicating that the antisera could neutralize the infection of FAdV on LMH cells.The recombinant proteins of FAdV-2-Fiber2,FAdV-4-Fiber2 and FAdV-8b-Fiber2 were extracted by ultrasonic crushing and immunized with Kunming white mice to prepare mouse polyclonal antibodies against the recombinant Fiber2 protein.Indirect immunofluorescence assay showed that the rat antiserum still had good activity and specificity when diluted at 1:200 times.In conclusion: Recombinant baculovirus with stable expression of Fiber2 protein was successfully constructed in this study,which further confirmed that the expressed Fiber2 protein has good immunogenicity.These results lay a solid foundation for the study of the biological function of Fiber2 protein of FAdV,and also provide scientific reference for the prevention and control of FAdV.更多还原