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.
作者
赵国欢
张小兵
李凡
李为民
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)
出处
《云南农业大学学报（自然科学版）》
CSCD
北大核心
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)
