犬瘟热病毒H蛋白基因重组腺病毒的构建及免疫效果评估

doi:10.13523/j.cb.20140110

中国生物工程杂志

2014, Vol. 34

Issue (1): 64-70 DOI: 10.13523/j.cb.20140110

技术与方法

犬瘟热病毒H蛋白基因重组腺病毒的构建及免疫效果评估

袁颖烁^1,2, 宋菲菲^1,2, 刘国强^1,2, 刘晔², 张守峰², 张乐萃¹, 扈荣良²

1. 青岛农业大学动物科技学院青岛 266109;
2. 军事医学科学院军事兽医研究所省部共建吉林省人兽共患病预防与控制重点实验室长春 130122

Construction and Immune Efficacy of Recombinant Adenovirus Expressing H Gene of Canine Distemper Virus

YUAN Ying-shuo^1,2, SONG Fei-fei^1,2, LIU Guo-qiang^1,2, LIU Ye², ZHANG Shou-feng², ZHANG Le-cui¹, HU Rong-liang²

1. Qingdao Agriculture University, College of Animal Science and Technology, Qingdao 266109, China;
2. Jilin Provincial Key Laboratory of Zoonosis Prevention and Control, and Military Veterinary Research Institute, Academy of Military Medical Sciences, Changchun 130122, China

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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。

关键词： 犬瘟热病毒; H基因; 腺病毒; 免疫效果

Abstract: The recombinant human adenovirus type 5 that expressed hemagglutinin(H) protein gene of canine distemper virus(CDV) be constructed. The shuttle and backbone plasmid were respectively constructed by reverse transcription polymerase chain reaction (RT-PCR) and the restriction enzyme digestion and ligation, 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.

Key words: Canine distemper virus Hemagglutinin gene Adenovirus Immune efficacy

收稿日期: 2013-03-25 出版日期: 2014-01-25

ZTFLH:

Q939.4

基金资助: 国家公益性行业（农业）科研专项经费资助项目（201203056）

通讯作者: 张乐萃，E-mail：lczhang@qau.edu.cn E-mail: lczhang@qau.edu.cn

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引用本文:

袁颖烁, 宋菲菲, 刘国强, 刘晔, 张守峰, 张乐萃, 扈荣良. 犬瘟热病毒H蛋白基因重组腺病毒的构建及免疫效果评估[J]. 中国生物工程杂志, 2014, 34(1): 64-70.

YUAN Ying-shuo, SONG Fei-fei, LIU Guo-qiang, LIU Ye, ZHANG Shou-feng, ZHANG Le-cui, HU Rong-liang. Construction and Immune Efficacy of Recombinant Adenovirus Expressing H Gene of Canine Distemper Virus. China Biotechnology, 2014, 34(1): 64-70.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140110 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I1/64

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