禽呼肠孤病毒σB和σC蛋白在昆虫细胞中的共表达

doi:10.16431/j.cnki.1671-7236.2020.05.006

摘要/Abstract

摘要： 本研究旨在获得具有天然构象和活性良好的禽呼肠孤病毒（ARV）σB和σC蛋白，采用杆状病毒表达系统在昆虫细胞中共表达σB和σC蛋白，并对其活性进行鉴定。根据NCBI数据库中ARV σB和σC基因序列设计引物，将两个基因克隆至pFast-Dual载体，转化大肠杆菌DH10Bac感受态细胞，筛选获得重组穿梭杆粒。通过转染sf9细胞，筛选到含有σB和σC基因的重组病毒。将重组病毒感染sf9细胞，通过优化接毒量和收获时间等参数，确定最佳表达条件，在sf9细胞中高效共表达σB和σC蛋白。Western blotting和间接免疫荧光（IFA）检测结果表明，成功在sf9细胞中共表达了ARV σB和σC蛋白，并具有很好的生物学活性。本试验结果为开发鉴别诊断试剂以及ARV颗粒疫苗的研究奠定了基础。

关键词: 禽呼肠孤病毒(ARV); σB基因; σC基因; 杆状病毒表达系统; sf9细胞

Abstract: To obtain the native conformation and antigenicity of σB and σC proteins of avian reovirus,the σB and σC proteins was expressed by Bac-to-Bac baculovirus expression system,and its reactivity was identified.The σB and σC genes of avian reovirus were amplified by designing two pairs of primers according to the sequence published on NCBI,inserted into donor plasmid pFast-dual to obtain recombinant donor plasmid,then transformed into E.coli DH10Bac,and the recombinant bacmid plasmid was obtained.Recombinant baculovirus was transfected into sf9 insect cells using cellfection reagent to obtain the production of recombinant viral contained the sequence of σB and σC genes.sf9 insect cells were inoculated by recombinant viral to explore the MOI and harvest timing expression condition,and the expression condition was optimized.The results of Western blotting and indirect immunofluorescence assay (IFA) showed that the recombinant σB and σC proteins were expressed successfully in sf9 insect cells and had good biological activity.This study provided a further research direction of ARV vaccine of virus-like particles and ARV subtype antibody ELISA detection kit.

Key words: avian reovirus (ARV); σB gene; σC gene; baculoviruses expression system; sf9 cell

中图分类号:

S831.7

王盛, 谢芝勋, 沈文康, 谢丽基, 范晴, 罗思思, 张艳芳, 曾婷婷, 黄娇玲, 张民秀, 谢志勤, 邓显文. 禽呼肠孤病毒σB和σC蛋白在昆虫细胞中的共表达[J]. 中国畜牧兽医, 2020, 47(5): 1334-1341.

WANG Sheng, XIE Zhixun, SHEN Wenkang, XIE Liji, FAN Qing, LUO Sisi, ZHANG Yanfang, ZENG Tingting, HUANG Jiaoling, ZHANG Mingxiu, XIE Zhiqin, DENG Xianwen. Expression of σB and σC Proteins of Avian Reovirus in Insect Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1334-1341.

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