柑桔黄龙病菌亚洲种菌毛形成蛋白基因序列分析、原核表达及抗血清制备

Cloning and prokaryotic expression of pilus formation protein gene from Candidatus Liberibacter asiaticus and preparation of its antiserum

柑桔黄龙病菌菌毛形成蛋白(pilus formation protein, 408)是一种丰度较高的分泌蛋白。以感染柑桔黄龙病(HLB)的海南琼海市绿橙叶片为材料提取总DNA,利用408基因的特异引物进行PCR扩增,获得该基因的目的片段。序列分析结果表明海南琼海黄龙病菌408基因与柑桔黄龙病菌亚洲种psy62株系 (GenBank登录号:CP001677.5)408基因序列一致。功能预测结果表明其含有一个与菌毛形成相关的N端结构域,以及C端含有两个高度保守的基序(Motif 1和Motif 2)。通过Eco R Ⅴ和Xho Ⅰ双酶切构建重组载体p ET32a-408并将其转化BL21(DE3)大肠杆菌。重组菌经终浓度为1 mmoL/L IPTG诱导,目的蛋白主要以包涵体形式表达。目的蛋白经Ni2+-NTA层析柱纯化,并以此为抗原,腹腔免疫小白鼠,获得效价在1∶500～1∶10000的多克隆抗血清;Western blot进一步分析表明,408蛋白多克隆抗血清特异性强。研究结果为柑桔黄龙病菌408蛋白功能研究和柑桔黄龙病菌的蛋白检测产品开发提供重要科学依据。

Pilus formation protein(408)is a secreted protein involved in the assembly of bacterial pili with high-level expression.The total DNA was extracted from the HLB-infected green orange leaves of Qionghai City,Hainan Province,China.The target fragment of408gene was obtained by PCR amplification with specific primers and sequence analysis showed that the408gene is identical with the homologue gene of the Candidatus Liberibacter asiaticus psy62isolate(GenBank accession number:CP001677.5).Functional prediction indicated that the protein contains a domain associated with Pilus formation protein at N terminal,while its C terminal contains two conserved motifs,Motif1and Motif2.The recombinant vector pET32a-408was constructed by using EcoRⅤand XhoⅠrestriction enzyme sites.The pET32a-408vector was transformed into E.coli BL21(DE3)and recombinant strain was induced by a final concentration of1mmoL/L IPTG.The408fusion protein was mainly expressed in the form of inclusion bodies.The408fusion protein was purified by Ni2+-NTA column and used to immunize mice by intraperitoneal injection.The408polyclonal antiserum was obtained and the titer was1:500~1:10000.Western blotting further analysis showed that408polyclonal antiserum was specific to408protein.This study provides a research basis for the function study of408protein and the development of protein detection products for HLB.