摘要
柑橘黄龙病菌CpaB基因编码一种菌毛组装蛋白(Flp pilus assemble protein),前期研究已经证实该蛋白具有外分泌性.在本研究中,首先构建pET30a-CpaB重组载体,然后将其转化BL21(DE3)感受态细胞,重组菌在37℃,0.2 mmol·L^(-1) IPTG诱导下,CpaB蛋白可被成功诱导表达,经过Ni-NTA亲和柱纯化后,获得纯度较高的CpaB重组蛋白,分子量为28.2 kDa,以其为抗原,免疫新西兰大白兔,制备多克隆抗血清;采用Western blot对多抗血清的特异性进行分析,结果显示该多抗能特异性结合纯化的CpaB蛋白;进一步使用制备好的CpaB抗血清对感染黄龙病和健康的柑橘样品进行DTBIA检测,具有较好的区分度.这些研究结果为柑橘黄龙病快速检测体系的建立提供理论参考.
CpaB of'Candidatus Liberibacter asiaticus'(CLas)is a Flp pilus assembly protein.Previous studies have proved that the protein is a secreted protein.The objective this study is prokaryotic expression the CpaB protein and preparation of its antiserum which would be used for C Las rapid detection.The recombinant vector pET30a-CpaB which linked with CpaB gene was transformed into E.coli BL21(DE3).CpaB protein was successfully induced at 37℃and by 0.2 a final concentration of 0.2 mmol·L^(-1) IPTG.The target protein was purified by Ni-NTA affinity column and used as antigen to prepare polyclonal antiserum.The specificity of polyclonal antibody serum analyzed by Western blot showed that the antiserum had certain specificity in binding with CpaB protein.The DTBIA result indicated that the antiserum has good discrimination between HLB infected citrus samples and healthy citrus samples.This study provide an important reference for the development of rapid detection production of Citrus Huanglongbing.
作者
顾佩佩
王莹
黄爱军
GU Peipei;WANG Ying;HUANG Aijun(School of Life Science,Gannan Normal University;National Navel Orange Engineering Research Center,Ganzhou 34100,China)
出处
《赣南师范大学学报》
2021年第6期74-77,共4页
Journal of Gannan Normal University
基金
江西省现代农业科研协同创新专项[JXXTCX2015002(3+2)-003]
江西省重点研发计划项目(20161ACF60016)
江西省现代农业产业技术体系建设专项(JXARS-07)
江西省重大科技研发专项(20194ABC28007)。
关键词
柑橘黄龙病
CpaB
原核表达
抗体制备
citrus Huanglongbing
CpaB
pokaryotic expression
antiserum preparation
