羊口疮病毒F1L、B2L基因重组腺病毒构建及对小鼠的免疫效果分析

doi:10.11843/j.issn.0366-6964.2018.08.015

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (8): 1701-1708.doi: 10.11843/j.issn.0366-6964.2018.08.015

羊口疮病毒F1L、B2L基因重组腺病毒构建及对小鼠的免疫效果分析

陈晓^1,2,3, 葛雷^1,3, 戴建军^1,3*, 李丽^2*, 张德福^1,3, 吴彩凤^1,3, 张树山^1,3

1. 上海市农业科学院畜牧兽医研究所, 上海 201106;
2. 锦州医科大学畜牧兽医学院, 锦州 121001;
3. 上海市农业遗传育种重点实验室动物遗传工程研究室, 上海 201106

收稿日期:2018-01-19 出版日期:2018-08-23 发布日期:2018-08-23
通讯作者: 戴建军,E-mail:blackman0520@126.com;李丽,E-mail:rainbow1992@163.com
作者简介:陈晓(1992-),女,辽宁锦州人,硕士生,主要从事预防兽医学研究,E-mail:550373412@qq.com
基金资助:
上海市科委实验动物专项（15140900300；17140900100）；上海市农委青年人才计划（沪农青字（2015）第1-36号）；上海市农业科学院攀高计划（PG171）；上海市种业发展项目（沪农科种字（2017）第3-3号）

Construction of Recombinant Adenovirus with Orf Virus F1L and B2L Genes and Analysis of Its Immune Efficacy in Mice

CHEN Xiao^1,2,3, GE Lei^1,3, DAI Jian-jun^1,3*, LI Li^2*, ZHANG De-fu^1,3, WU Cai-feng^1,3, ZHANG Shu-shan^1,3

1. Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;
2. College of Animal Husbandry & Veterinary, Jinzhou Medical University, Jinzhou 121001, China;
3. Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-genetics and Breeding, Shanghai 201106, China

Received:2018-01-19 Online:2018-08-23 Published:2018-08-23

摘要/Abstract

摘要：

选取自剪切T2A和P2A肽，连接F1L和B2L基因，构建羊口疮病毒（orf virus，ORFV）重组腺病毒，并对其免疫效果进行评价，为羊口疮的疫苗研制提供依据。应用PCR扩增ORFV-F416毒株的F1L、B2L目的基因，构建重组腺病毒穿梭质粒，穿梭质粒与骨架质粒共转染HEK-293细胞，包装出ORFV重组腺病毒。将重组腺病毒感染MDBK细胞，收集上清液免疫ICR小鼠，并进行间接ELISA、淋巴细胞增殖、ORFV攻毒试验等初步评价小鼠免疫效果。结果显示：成功获得pAV-CMV-F1L-T2A-B2L-P2A-EGFP重组腺病毒，其TCID₅₀为10^-5.375·mL^-1，PCR和IFA检测证实目的基因在基因及蛋白水平上均有表达。ORFV重组腺病毒免疫小鼠后，其抗体水平迅速升高。ORFV重组腺病毒组对特异性抗原ORFV表现出明显的T淋巴细胞增殖能力，极显著高于PBS组（P<0.01）。本文的研究结果表明：F1L和B2L基因重组腺病毒能够刺激小鼠产生体液免疫和细胞免疫应答，且未影响小鼠正常采食，保护效力较好。

Abstract:

The recombinant adenoviruses of orf virus (ORFV) was constructed by splicing F1L and B2L genes with the self-cut T2A and P2A peptide, and its immune effects were evaluated, which provided the basis for vaccine development of ORFV. The target genes of F1L and B2L from ORFV-F416 strain were amplified by PCR, and the recombinant adenovirus shuttle plasmid was constructed. The shuttle plasmid and backbone plasmid were co-transfected into HEK-293 cells and the ORFV recombinant adenovirus was packaged. The recombinant adenovirus was used to infect MDBK cells and the supernatant was collected to immunize ICR mice. Indirect ELISA, lymphocyte proliferation and ORFV challenge were conducted to evaluate the immunogenicity of mice. The results showed that pAV-CMV-F1L-T2A-B2L-P2A-EGFP recombinant adenovirus was successfully obtained, and its TCID₅₀ was 10^-5.375·mL^-1. PCR and IFA results showed that the target gene was expressed both in gene and protein levels. After immunization with ORFV recombinant adenovirus, the antibody level of mice increased rapidly. ORFV recombinant adenovirus group showed much higher ability of T lymphocyte proliferation, which was significantly higher than that of PBS group (P<0.01). The results of this article showed that the recombinant adenovirus of F1L and B2L genes had positive protective effect in mice, which could stimulate humoral and cellular immune responses, and have no effects on feed intake.

中图分类号:

S852.659.1

陈晓, 葛雷, 戴建军, 李丽, 张德福, 吴彩凤, 张树山. 羊口疮病毒F1L、B2L基因重组腺病毒构建及对小鼠的免疫效果分析[J]. 畜牧兽医学报, 2018, 49(8): 1701-1708.

CHEN Xiao, GE Lei, DAI Jian-jun, LI Li, ZHANG De-fu, WU Cai-feng, ZHANG Shu-shan. Construction of Recombinant Adenovirus with Orf Virus F1L and B2L Genes and Analysis of Its Immune Efficacy in Mice[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1701-1708.

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羊口疮病毒F1L、B2L基因重组腺病毒构建及对小鼠的免疫效果分析

Construction of Recombinant Adenovirus with Orf Virus F1L and B2L Genes and Analysis of Its Immune Efficacy in Mice

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