柑橘黄龙病病原CLIBASIA＿03230基因的克隆及其编码蛋白的原核表达  

The Cloning and Prokaryotic Expression of Candidatus Liberibacter Asiaticus CLIBASIA＿03230 Gene

作　　者：赵国欢 张小兵[3] 李凡[1] 李为民[2] ZHAO Guohuan;ZHANG Xiaobing;LI Fan;LI Weimin(Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China,Yunnan Agricultural University,Kunming 650201,China;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China;Biology Institue,Hebei Academy of Sciences,Shijiazhuang 050051,China)

机构地区：[1]云南农业大学农业生物多样性与病虫害控制教育部重点实验室,云南昆明650201 [2]中国农业科学院生物技术研究所,北京100081 [3]河北省科学院生物研究所,河北石家庄050051

出　　处：《云南农业大学学报（自然科学版）》2019年第1期9-14,共6页Journal of Yunnan Agricultural University:Natural Science

基　　金：中国农业科学院科技创新工程协同创新任务(CAAS-STCX2016013);中央级公益性科研院所基本科研业务费专项(Y2016XT04)

摘　　要：【目的】柑橘黄龙病(Citrus Huanglongbing,HLB)是由韧皮部杆菌(Candidatus Liberibacter spp.)引发的重要柑橘病害。本试验利用生物信息学方法分析韧皮部杆菌全基因组,推测CLIBASIA_03230基因(命名为Clsp11)编码分泌蛋白,克隆该基因并构建其原核表达载体,表达、纯化Clsp11蛋白,以用于制备特异抗体。【方法】利用CTAB法从感染柑橘黄龙病病原的长春花中提取总DNA,以此为模板经PCR扩增Clsp11,并克隆至pMD18-T载体,挑取阳性克隆测序验证;利用无缝克隆技术将Clsp11克隆至原核表达载体pET30a,通过PCR和酶切鉴定筛选阳性克隆,命名为pET30a-Clsp11;转化大肠杆菌BL21,经IPTG诱导,表达N-端融合6×His标签的Clsp11重组蛋白,SDS-PAGE电泳检测,分析目的蛋白的表达水平,并利用镍柱进行纯化。【结果】Clsp11蛋白成功表达,IPTG诱导5 h表达量显著提高,主要以包涵体形式存在于宿主菌中。【结论】本研究克隆了柑橘黄龙病病原Clsp11基因,构建了原核表达载体pET30a-Clsp11,并成功表达、纯化了Clsp11重组蛋白,为进一步制备特异性抗体和研究Clsp11生物学功能奠定了基础。[Purpose]Pathogenic bacteria secreted proteins play an important role in the process of pathogen infection.Analysis of the biological functions of secretory proteins will help to clarify the molecular mechanism of pathogen-plant interaction.In this study,the entire genome of Candidatus Liberibacter asiaticus was subjected to bioinformatics analysis,and CLIBASIA_03230 gene(named as Clsp11)was predicted to encode a secreted protein.Therefore,the Clsp11 was cloned and inserted into the prokaryotic expression vector,and the Clsp11 protein was expressed and purified for preparation of the specific antibodies in the next step.[Method]Total DNA was extracted from Ca.L.asiaticus-infected periwinkle,and was used as template to amplify Clsp11.The resulting PCR products were cloned into pMD18-T vector and sequenced.With the In-Fusion cloning technique,the Clsp11 was inserted into the pET30a,resulting in pET30a-Clsp11.The pET30a-Clsp11 was subsequently transformed into Escherichia coli BL21 and induced by IPTG to express the recombinant protein Clsp11,the N-terminus of which was fused with 6×His tag.After analysis with SDS-PAGE,the Clsp11 recombinant protein was purified by nickel column.[Result]The Clsp11 protein was successfully expressed,and reached high expression level when IPTG induction for 5 hours.Additionally,a large portion of the recombinant proteins present as inclusion bodies.[Conclusion]In this study,the Clsp11 of Ca.L.asiaticus was cloned,the prokaryotic expression vector pET30a-Clsp11 was constructed,and the recombinant protein Clsp11 was expressed and purified,thus laying the foundation to prepare specific antibodies as well as investigate the biological function(s)of Clsp11.

关 键 词：柑橘黄龙病 Clsp11 基因克隆 原核表达 

分 类 号：S436.661.19[农业科学—农业昆虫与害虫防治]