摘要
【目的】本研究旨在明确中华蜜蜂Apis cerana cerana热休克蛋白70(Heat shock protein 70,HSP70)与中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)的关系。【方法】首先根据NCBI上已公布的中华蜜蜂HSP70的全基因(NO.MH122655.1)序列,设计特异性引物,提取3日龄中华蜜蜂幼虫总RNA,通过RT-PCR方法获得HSP70基因,并通过生物信息学软件进行分析;其次将其克隆到原核表达载体pET-32a中,转化入大肠杆菌BL21(DE3)进行蛋白表达,并制备重组蛋白,将纯化的重组蛋白免疫小鼠制备多克隆抗体;最后利用制备多克隆抗体作为一抗,通过Western-blot方法检测CSBV感染中华蜜蜂前后HSP70在幼虫体内的变化。【结果】成功获取中华蜜蜂HSP70蛋白的全基因,其核苷酸序列全长为1833 bp,生物信息学分析后,发现其编码蛋白存在7个抗原表位。利用原核表达系统,成功获得大小约87 ku的重组蛋白HSP70,免疫小鼠后获得多克隆抗体,经Western-blot分析能与标签蛋白发生特异性反应;对感染CSBV前后蜜蜂体内HSP70蛋白检测发现感染后HSP70表达水平显著提高。【结论】通过原核表达系统,成功表达中华蜜蜂HSP70基因和HSP70多克隆抗体,并证实CSBV感染后影响中华蜜蜂体内HSP70蛋白表达量的改变,为深入研究HSP70功能提供了帮助。
[Objectives]To clarify the relationship between Apis cerana cerana heat shock protein 70(HSP70)and Chinese sacbrood virus(CSBV).[Methods]A specific pair of primers were developed for HSP70 based on the complete A.cerana cerana genome listed in NCBI(NO.MH122655.1).The HSP70 gene was amplified from 1-3 day old A.cerana cerana by RT-PCR using extracted total RNA as the template,then analyzed with bioinformatics tools.The resultant HSP70 was cloned into the prokaryotic expression vector pET-32 a,then transformed into Escherichia coli BL21(DE3)for protein expression.The resultant recombinant protein was purified by affinity chromatography and the purified,recombinant,fusion protein was then injected into mice to create polyclonal antibodies as primary antibodies.Changes in the expression of HSP70 in larvae before,and after,CSBV infection of A.cerana cerana were detected using the Western-blot method.[Results]A 1833 bp HSP70 gene was successfully cloned,and a recombinant plasmid(pET-32 a-HSP70)correctly constructed.Bioinformatics analysis revealed seven epitopes in the protein.Using a prokaryotic expression system,we obtained a recombinant fusion protein of HSP70 with a size of about 87 ku,which was purified and injected into mice to obtain polyclonal antibodies.Western-blot analysis indicated specific reactions with the tag protein.HSP70 expression significantly increased after CSBV infection.[Conclusion]The HSP70 gene and HSP70 polyclonal antibody were successfully expressed through a prokaryotic expression system.HSP70 protein expression in the Chinese honeybee is affected by CSBV infection,a finding that promotes further research on the function of HSP70.
作者
岳金金
马跃宇
王琛
张溪研
费东亮
马鸣潇
YUE Jin-Jin;MA Yue-Yu;WANG Chen;ZHANG Xi-Yan;FEI Dong-Liang;MA Ming-Xiao(Jinzhou Medical University,Jinzhou 121000,China)
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2021年第5期1057-1064,共8页
Chinese Journal of Applied Entomology
基金
国家自然基金面上项目(31772760)
国家自然基金面上项目(31972626)。
关键词
中华蜜蜂
热休克蛋白70
多克隆抗体
蛋白纯化
中蜂囊状幼虫病毒
Apis cerana cerana
heat shock protein 70
polyclonal antibody
protein purification
Chinese sacbrood virus
