慢性蜜蜂麻痹病毒SP1蛋白的原核表达及免疫原性分析
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摘要
慢性蜜蜂麻痹病毒(Chronic bee paralysis virus,CBPV)主要侵袭成年工蜂,造成患病蜜蜂神经麻痹,丧失飞行能力,出现爬行现象。CBPV据推测有两个结构蛋白SP1和SP2,通过对SP1蛋白的免疫原性探索,为SP1蛋白的功能研究和CBPV生物制剂研制奠定基础。本研究根据大肠杆菌密码子的偏嗜性,对CBPVSP1基因进行优化和合成,将其克隆至原核表达载体pET-32a,获得重组表达质粒pET-Opti-SP1。将重组质粒转化至宿主菌BL21(DE3)中诱导表达,用SDS-PAGE和Western Blot方法对该蛋白进行分析和鉴定,然后对最佳表达条件进行摸索。在此基础上,用纯化后的SP1蛋白免疫接种BALB/c小鼠,同时设置纯化病毒组、pET-32a对照组和空白对照组,检测免疫小鼠的淋巴细胞增殖指数(SI)和血清中抗体水平。结果表明,首次成功表达和纯化了重组SP1蛋白,且该蛋白能诱导产生特异性抗CBPV的抗体,促进淋巴细胞的增殖和提高IL-4、IL-10、IFN-γ水平,具有较好的免疫原性。本研究为进一步研究CBPV SP1蛋白的功能和防治CBPV生物制剂的研发提供了帮助。
Chronic bee paralysis virus(CBPV) mainly invades adult worker bees, causing nerves paralyzed by sick bees, losing flight ability and crawling. It is speculated that there are two structural proteins SP1 and SP2 in CBPV.The immunogenicity of the SP1 protein was explored. We wished to lay a foundation for the functional study of the SP1 protein and development of biologic agents against the CBPV. The SP1 gene of the CBPV was optimized and synthesized according to a preference for the Escherichia coli codon. The SP1 gene was cloned into the pET-32 a vector, and the recombinant prokaryotic expression plasmid pET-Opti-SP1 was obtained. The recombined plasmids were transformed into BL21(DE3) to induce expression, respectively. Proteins were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot, and then the optimal expression conditions were explored. On this basis, BALB/c mice were immunized with purified SP1 protein. A "pure virus" group, pET-32 a group, and a blank control group were created. Then, the antibody level, lymphocyte proliferation index, as well as levels of interleukin(IL)-4, IL-10 and interferon(IFN)-γ in the immunized mice were determined. We first showed that the recombinant SP1 protein was expressed and purified. The recombinant SP1 protein could induce production of specific antibodies against it, promote lymphocyte proliferation, increase levels of IL-4, IL-10, and IFN-γ, and have high immunogenicity. The present study provided insights for functional studies of the SP1 protein of the CBPV, as well as development of biologic agents for the prevention and treatment of CBPV infection.
Chronic bee paralysis virus(CBPV) mainly invades adult worker bees, causing nerves paralyzed by sick bees, losing flight ability and crawling. It is speculated that there are two structural proteins SP1 and SP2 in CBPV.The immunogenicity of the SP1 protein was explored. We wished to lay a foundation for the functional study of the SP1 protein and development of biologic agents against the CBPV. The SP1 gene of the CBPV was optimized and synthesized according to a preference for the Escherichia coli codon. The SP1 gene was cloned into the pET-32 a vector, and the recombinant prokaryotic expression plasmid pET-Opti-SP1 was obtained. The recombined plasmids were transformed into BL21(DE3) to induce expression, respectively. Proteins were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot, and then the optimal expression conditions were explored. On this basis, BALB/c mice were immunized with purified SP1 protein. A "pure virus" group, pET-32 a group, and a blank control group were created. Then, the antibody level, lymphocyte proliferation index, as well as levels of interleukin(IL)-4, IL-10 and interferon(IFN)-γ in the immunized mice were determined. We first showed that the recombinant SP1 protein was expressed and purified. The recombinant SP1 protein could induce production of specific antibodies against it, promote lymphocyte proliferation, increase levels of IL-4, IL-10, and IFN-γ, and have high immunogenicity. The present study provided insights for functional studies of the SP1 protein of the CBPV, as well as development of biologic agents for the prevention and treatment of CBPV infection.
引文
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[10]钟义.慢性蜜蜂麻痹病毒Ch1株编码区的序列分析与半套式RT-PCR检测方法的建立[D].锦州医科大学,2016,18.
[11]Bailey L,Gibbs A,Woods R.Two viruses from adult honey bees(Apis mellifera Linnaeus)[J].Virology,1963,21(3):390-395.
[12]冯峰,陈淑静.慢性蜜蜂麻痹病的分布危害及季节特征和防治药物[J].中国养蜂,1990,(1):5-6.
[13]吴艳艳,周婷,王强,代平礼.蜜蜂慢性麻痹病的防治[J].中国蜂业,2014,65(5):23-24.
[2]Wilfert L,Long G,Leggett H C.Deformed wing virus is a recent global epidemic in honeybees driven by Var-roa mites[J].Science.2016,351(6273):594-597.
[3]Amiri E,Meixner M,Büchler R,Kryger P.Chronic bee paralysis virus in honeybee queens:evaluating sus-ceptibility and infection routes[J].Viruses,2014,6(3):1188-1201.
[4]Chevin A,Coutard B,Blanchard P,Dabert-Gay A S,Cha-bert M R,Théiry R.Characterisation of structural proteins from chronic bee paralysis virus(CBPV)using mass spec-trometry[J].Viruses,2015,7(6):3329-3344.
[5]Tantillo G,Bottaro M,Di Pinto A,Martella V,Di Pin-to P,Terio V.Virus infections of honeybees Apis Mellifera[J].Ital J Food Saf,2015,4(3):5364.
[6]Desai S D,Currie R W.Effects of wintering environ-ment and parasite-pathogen interactions on honey bee colony loss in north temperate regions[J/OL].PLoSOne,2016,11(7):e0159615.
[7]Elbeaino T,Daher-Hiaii N,Ismaeil F,Mando J,Khaled B S,Kubaa R A.Occurrence of deformed wing virus,chronic bee paralysis virus and mtDNA variants in haplotype K of varroa destructor mites in Syrian apiaries[J].Exp Appl Acarol,2016,69(1):11-19.
[8]Tseveqmid K,Neumann P,Ya?ez O.The honey bee pa-thosphere of Mongolia:European Viruses in Central Asia[J/OL].PLoS One,2016,11(3):e0151164.
[9]汪天澍,施腾飞,刘芳,余林生,齐磊,孟祥金.安徽省七种蜜蜂病毒的发生与流行研究[J].应用昆虫学报,2015,52(2):324-332.
[10]钟义.慢性蜜蜂麻痹病毒Ch1株编码区的序列分析与半套式RT-PCR检测方法的建立[D].锦州医科大学,2016,18.
[11]Bailey L,Gibbs A,Woods R.Two viruses from adult honey bees(Apis mellifera Linnaeus)[J].Virology,1963,21(3):390-395.
[12]冯峰,陈淑静.慢性蜜蜂麻痹病的分布危害及季节特征和防治药物[J].中国养蜂,1990,(1):5-6.
[13]吴艳艳,周婷,王强,代平礼.蜜蜂慢性麻痹病的防治[J].中国蜂业,2014,65(5):23-24.