摘要
为了检测中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)抗体水平,用Bac-to-Bac杆状病毒表达系统,构建含有CSBV VP2的重组杆状病毒rBac-VP2,并在昆虫细胞中获得表达。用经过纯化的重组VP2蛋白作为包被抗原,建立CSBV抗体的间接酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)检测方法。结果表明成功的表达了VP2蛋白。优化反应条件后,建立的ELISA检测方法仅与CSBV阳性IgY发生特异性反应,与其它蜜蜂病毒的阳性IgY不发生交叉反应,且检测阳性IgY的灵敏度达到了1∶6 400,证明其具有良好的特异性和敏感性。批内重复与批间重复变异系数均小于10%。与CSBV全病毒包被进行比较,检测结果差异不显著,说明该方法适用于抗CSBV抗体检测。
To determine the level of antibody against the Chinese Sacbrood Virus(CSBV),the recombinant baculovirus rBac-CSBV VP2 gene was constructed. The expressed VP2 protein in insect cells was obtained using the Bac-to-Bac?baculovirus expression system. An indirect enzyme-linked immunosorbent assay(ELISA)was developed to detect anti-CSBV antibody using the purified VP2 protein as the coating antigen. The VP2 protein was expressed successfully. After optimization of reaction conditions,ELISA was highly specific to immunoglobulin(Ig)Y of the CSBV with no evidence of cross-reactivity with the IgY of other bee viruses. The sensitivity of detection of IgY reached 1∶6,400 dilution,which showed that ELISA had good specificity and sensitivity. The coefficients of variation within and between batches were <10%. Compared with the virus coating of the whole CSBV,there was no significant difference in detection. The described method appears to be suitable for the detection of anti-CSBV antibody.
引文
[1] Reddy K E,Yoo M S,Kim Y H,Kim N H,Ramya M,Jung H N,Thao L T B,Lee H S,Kang S W. Ho-mology differences between complete sacbrood virus ge-nomes from infected Apis mellifera and Apis cerana hon-eybees in Korea[J]. Virus Genes,2016,52(2):281-289.
[2] Tsevegmid K,Neumann P,Ya?ez O. The honey bee pathosphere of mongolia:European viruses in central Asia[J/OL]. PLoS One,2016,11(3):e0151164.
[3] Nguyen N,Le T. Complete genome sequence of sac-brood virus strain SBM2,isolated from the honeybee Apiscerana in vietnam[J/OL]. Genome Announc,2013,1(1):pii:e00076-12.
[4] Hu Y,Fei D L,Jiang L L,Wei D,Li F B,Diao Q Y,Ma M X. A comparison of biological characteristics of three strains of Chinese sacbrood virus in Apis cerana[J].Sci Rep,2016,6:37424.
[5] Bailey L. Viruses attacking the honey bee[J].Adv Virus Res,1976,20:271-304.
[6] Ghosh R C,Ball B V,Willcocks M M,Carter M J.The nucleotide sequence of sacbrood virus of the honey bee:an insect picorna-like virus[J]. Gen Virol,1999,80(6):1541-1549.
[7] Sun L,Li M,Fei D L,Diao Q Y,Wang J,Li L Q,Ma M X. Preparation and application of egg yolk antibod-ies against Chinese sacbrood virus infection[J]. Front Microbiol,2018,9:1814.
[8] Fei D L,Zhang H C,Diao Q Y,Jiang L L,Wang Q,Fan Z B,Ma M X. Codon optimization expression in escherichia coli,and immunogenicity of recombinant Chi-nese sacbrood virus(CSBV)structural proteins VP1,VP2,and VP3[J/OL]. PLoS One,2015,10(7):e0134423.
[9]李芳兵,费东亮,张皓淳,胡影,魏东,张鹤,岳金金,马鸣潇,曲祖乙.中蜂囊状幼虫病毒结构蛋白VP2的密码子优化表达及其免疫原性研究[J].病毒学报,2017,33)2:231-237. DOI:10.13242/j.cnki.bingduxuebao.003127
[10]Luckow V A,Lee S C,Barry G F,Olins P O. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli[J]. J Virol,1993,67(8):4566-4579.
[11]杨嘉玉,韦莉,王菁,侯磊,全荣,朱珊珊,阎旭,李子璇,刘超,刘爵.猪流行性腹泻病毒N蛋白杆状病毒表达与间接ELISA方法建立[J].中国兽医杂志,2016,52(5):21-23.
[12]郑光来,卢晓冉,张静远,陈腾,王东方,严妍,张守峰,扈荣良.狂犬病病毒M蛋白在杆状病毒中的表达、纯化及多克隆抗体的制备[J].病毒学报,2016,32(4):472-477. DOI:10.13242/j.cnki.bingduxuebao.003127
[13]张立霞,周剑芳,于在江,舒跃龙.杆状病毒表面展示甲型流感病毒核蛋白的免疫原性及保护效果的初步评价[J].病毒学报,2013,29(3):265-272. DOI:10.13242/j.cnki.bingduxuebao.002385
[14]Miller L K. Baculoviruses for foreign gene expression in insect cells[J]. Biotechnology(Reading Mass),1988,10:457-465.
[15]Ailor E,Betenbaugh M J. Modifying secretion and post-translational processing in insect cells[J]. Curr Opin Bio-tech,1999,10(2):142-145.
[16]Hitchman R B,Possee R D,King L A. Baculovirus ex-pression systems for recombinant protein production in insect cells[J]. Recent Pat Biotechnol,2009,3(1):46-54.
[17]冯峰.中国蜜蜂病理及防治学(第1版)[M].北京:中国农业科技出版,1995:67.
