摘要
研究选取犬瘟热病毒H蛋白基因为目的基因,利用反转录聚合酶链式反应(RT-PCR)、酶切、连接等方法构建出重组人5型腺病毒穿梭质粒和骨架质粒,两者共转染293AD细胞并包装出重组腺病毒。通过电镜负染、酶切鉴定、Western blot及一步生长曲线测定等方法进行病毒的生物学特性鉴定,证明犬瘟热病毒H蛋白基因重组腺病毒构建正确。犬分别肌肉接种CDV疫苗毒和重组腺病毒,并检测免疫后第14,28,42,56,70,84d时犬血清中抗CDV中和抗体的变化情况。结果显示,犬免疫重组腺病毒后体内产生的抗CDV平均中和抗体水平高于对照组,滴度最高达2-8。研究表明,正确构建的重组腺病毒具有良好的免疫原性,诱导产生的抗犬瘟热病毒中和抗体达到犬最低免疫保护水平,为进一步研发犬瘟热病毒活载体疫苗提供理论依据。
by reverse transcription polymerase chain reaction （RT-PCR） and the restriction enzyme ctlgesuon ana llgauon, then co-transfected into the 293AD cell lines after Pac I linearization respectively. The biological characterization was determined by the electron microscope and restriction enzyme digestion and western blotting analysis and the one-step growth curve, the results proved that the recombinant human adenovirus type 5 that expressed H gene of CDV was constructed correctly. The anti-CDV antibody titers of the dog serum were detected by using neutralization test after a single intramuscular immunization with CDV vaccine strains or recombinants on days 14, 28, 42, 56, 70, 84. The results showed that average anti-CDV antibody titers （up to 2-8 ） were higher than that of the control group. It implied that the recombinant was constructed correctly and has a good immunogenicity, and it could stimulate an effective level of neutralizing antibodies which reached the lowest level of immune protection against CDV. This work provides a theoretical basis for the research and development of a more efficient distemper live vector vaccine.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2014年第1期64-70,共7页
China Biotechnology
基金
国家公益性行业(农业)科研专项经费资助项目(201203056)
关键词
犬瘟热病毒
H基因
腺病毒
免疫效果
Canine distemper virus Hemagglutinin gene Adenovirus Immune efficacy
