摘要
【目的】柑橘黄龙病(Citrus Huanglongbing,HLB)是柑橘产业的头号杀手,其病原主要是韧皮部杆菌属亚洲种(Candidatus Liberibacter asiaticus,CLas),为革兰氏阴性菌,田间主要经亚洲柑橘木虱(Diaphorina citri)传播,为深入研究该病菌分泌蛋白的功能,笔者筛选其中1个分泌蛋白,进行原核表达并制备抗体。【方法】设定4点条件从Prasad等预测的166个分泌蛋白基因中筛选,与pET-28a构建重组表达载体,以原核表达的融合蛋白作为抗原,用于注射兔子,制备多克隆抗体。【结果】从预测的166个分泌蛋白基因中筛选出05150基因,成功构建表达载体pET-28a-05150,表达菌株pET-28a-05150在18℃、0.1 mmol·L^-1 IPTG浓度条件下诱导的目的蛋白为包涵体。获得的pET-28a-05150抗血清通过ELISA效价为1∶4000,通过dot ELISA检测手段可与田间感病样品发生显色反应,通过Western blot能与融合蛋白杂交出特异性条带。【结论】该研究筛选了CLas的分泌蛋白基因05150,以其体外表达蛋白制备了pET-28a-05150抗血清,为研究05150基因功能奠定前期基础,也为建立HLB的血清学检测手段及探究柑橘黄龙病菌分泌蛋白基因的筛选方法提供参考。
【Objective】Citrus Huanglongbing(HLB)is the most destructive disease of citrus worldwide.In China,HLB is caused by Candidatus Liberibacter asiaticus(CLas),a gram-negative,phloemrestricted and psyllid-transmitted bacterium.The citrus industry is facing an unprecedented challenge.At present,the world’s difficult problem of pure cultivation of CLas has not been overcome.The indepth study on its physical and chemical characteristics and the development of disease management technologies are facing great challenges,and the research on the interaction between secreted proteins and citrus is not clear completely.In order to research the function of secreted proteins of CLas,one secreted protein was screened,expressed in vitro,and its polyclonal antibodies were made.【Methods】Based on the 166 predicted secreted protein genes of Prasad et al.,four-point screening conditions were set:1)the genes can be analyzed by using all the four softwares:Phbius,SigP4.1,LipoP,and SigP3.0;2)the predicted signal peptide of genes has secretory function that has been verified by alkaline phosphatase(PhoA);3)the protein size is between 10 ku and 25 ku;4)the expression of potential CLas secreted proteins in infected citrus can be determined by reverse transcription-polymerase chain reaction(RT-PCR).We amplified the secreted protein gene screened from infected samples collected from Jiangxi province by PCR.The expression vector with NdeⅠ(F)and XhoⅠ(R)restriction sites was successfully constructed for the screened secreted protein gene based on vector pET-28a,and the expression vector was transferred into Escherichia coli BL21(DE3).The expression strain was induced to express by the IPTG concentrations,which were set to 0,0.1,0.3,0.5,0.7 and 1.0 mmol·L^-1.Then the optimal induction IPTG concentration was selected to induce the expressed protein,and Western blot was addressed to verify whether the target protein was expressed using Anti-his-tag mAb and Anti-IgG(H+L chain)(Mouse)pAb-HRP.The purified fusion protein was approached as an antigen to immunize rabbits intraperitoneally to prepare polyclonal antibodies.Then direct binding of the antibody with the purified fusion protein was evaluated using indirect enzyme-linked immunosorbent assay(ELISA),ELISA plates were coated with 100μL of the purified fusion protein(1:4 dilutions in carbonate coating buffer),the concentrations of the polyclonal antibodies were set to 1∶500,1∶1000,1∶2000……1∶8192000 and 1∶16384000,respectively,and Anti-IgG(H+L chain)(Rabbit)pAb-HRP was diluted to 1∶8000 with 1×PBS buffer.Then dot ELISA and Western blot detection methods were addressed to verify the effectiveness of the polyclonal antibodies with the field samples.The concentrations of the polyclonal antibodies were set to 1∶1000,1∶3000,1∶5000,1∶7000,and 1∶10000,respectively.Anti-rabbit IgG(whole molecule)-AP or Anti-IgG(H+L chain)(Rabbit)pAb-HRP was diluted to 1∶8000 with 1×PBS buffer.Total protein was extracted by 1×PBS buffer from field samples collected in May,September,and November from Jiangxi,Sichuan,and Fujian provinces,respectively.【Results】In this study,six secreted protein genes were screened preliminarily from the 166 predicted secreted protein genes,and indirect verification of six secreted proteins expression was conducted in infected citrus from Jiangxi province by RT-PCR.We only amplified the 05150 sequence in infected citrus,which was highly conserved among six CLas strains whose genome sequences were available with 100%identity in nucleotide sequences.CLIBASIIA_05150 was screened,and the protein size was approximately 22 ku(i.e.,excluding the predicted N-terminal signal peptide,1-33 aa).The 05150 sequence was ligated to the vector pET-28a,and the expression vector pET-28a-05150 was successfully constructed,and pET-28a-05150 was transferred into E.coli BL21(DE3)successfully.The optimized induction for final IPTG concentration of the expression strain pET-28a-05150 was 0.1 mmol·L^-1,and fusion protein was insoluble in the mixed solution instead of the supernatant,indicating the formation of inclusion bodies.The pET-28a-05150 polyclonal antibody was evaluated for binding affinity to the pET-28a-05150 antigen by indirect ELISA.The binding affinity was tested using different concentrations of the polyclonal antibody,confirming the optimal concentration of pET-28a-05150 polyclonal antibody for indirect ELISA 1∶4000 and the sensitivity 1∶32000.Dot ELISA assured its color reaction with infected samples from the field.The positive dot ELISA detections accounted for 38.9%of the positive PCR detections,of which 4 and 2 samples were collected from Jiangxi province in September and May,respectively,1 from Sichuan province in September.Western blot assured that pET-28a-05150 polyclonal antibody hybridized with the fusion protein but not with infected citrus.【Conclusion】In this study,secreted protein gene 05150 of CLas was screened,and pET-28a-05150 antiserum was prepared with its expressed protein in vitro,which laid a preliminary foundation for studying the function of 05150,and also provided a reference for establishing serological detection and screening methods for secreted protein gene of HLB.
作者
晏建红
关巍
宾羽
黄洋
周常勇
赵廷昌
YAN Jianhong;GUANWei;BIN Yu;HUANG Yang;ZHOU Changyong;ZHAO Tingchang(Citrus Research Institute,Southwest University/National Citrus Engineering Research Center,Chongqing 400712,China;Institute of Plant Protection,Chinese Academy of Agricultural Sciences/State Key Laboratory for Biology of Plant Diseases and Insect Pests,Beijing 100193,China)
出处
《果树学报》
CAS
CSCD
北大核心
2020年第9期1384-1393,共10页
Journal of Fruit Science
基金
国家重点研发计划项目(2018YFD0201500)
中央本级重大增减支项目(2060302)
中国农业科学院科技创新工程协同创新任务(CAAS-STCX2016013)
国家自然科学基金(31701754)。
关键词
柑橘黄龙病
分泌蛋白基因
原核表达
抗血清制备
Citrus Huanglongbing
Secreted protein gene
Prokaryotic expression
Preparation of antiserum
作者简介
晏建红,女,硕士,研究方向:柑橘黄龙病。Tel:18875099040,E-mail:jianhong0903@163.com;通信作者:周常勇,E-mail:zhoucy@cri.cn;通信作者:赵廷昌,E-mail:zhaotgcg@163.com。
