柑橘黄龙病菌亚洲种菌毛装配蛋白基因(PAP)序列分析、原核表达及抗血清制备

Cloning and prokaryotic expression of Flp pilus assembly protein gene (PAP) from Candidatus Liberibacter asiaticus and preparation of antiserum against PAP

菌毛装配蛋白(Flp pilus assembly protein,PAP)是一种分泌蛋白,参与细菌菌毛的组装,表达量高。本研究以感染柑橘黄龙病的海南琼海市绿橙叶片为材料提取总DNA,用其菌毛装配蛋白基因(PAP)的特异引物进行PCR扩增,获得该基因的目的片段,序列分析表明海南琼海柑橘黄龙病菌PAP基因与柑橘黄龙病菌亚洲种(GenBank登录号:CP001677.5)PAP基因序列一致。功能预测表明它含有两个与分泌功能相关的CpaC和Secretin结构域。PCR产物通过Eco RⅤ和XhoⅠ双酶切构建重组载体pET32a-PAP。将pET32a-PAP载体转化BL21(DE3)大肠杆菌,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Ni^2+-NTA层析柱纯化,并作为抗原腹腔免疫小白鼠,获得效价在1∶500~1∶1 000的多克隆抗血清。Western blot分析表明,PAP多克隆抗血清特异性强;以田间样品总蛋白做抗原时,能特异性检测染病样品。本研究为PAP蛋白功能研究和开发柑橘黄龙病菌的蛋白检测产品提供研究基础。

Flp pilus assembly protein(PAP)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 collected from Qionghai city,Hainan province,China.The target fragment of PAP gene was obtained by PCR amplification with specific primers.Sequence analysis showed that the PAP gene is identical with the PAP gene of the Candidatus Liberibacter asiaticus(GenBank accession number:CP001677.5).Function prediction of PAP indicated that it contains two domains associated with secretory function,CpaC and Secretin.PCR products were digested by Eco RⅤand XhoⅠrestriction enzyme and cloned into vector pET32a.The recombinant pET32a-PAP vector was transformed into Escherichia coli BL21(DE3)and PAP was expressed in the form of inclusion bodies under induction by 1 mmoL/L IPTG.The PAP fusion protein was purified by Ni^2+-NTA column and used as an antigen.The mice were immunized intraperitoneally to obtain polyclonal antiserum and the titer was 1∶500-1∶1 000.Western blotting further analysis showed that PAP polyclonal antiserum was specific to PAP protein,which is applicable to field samples detection by ELISA.This study provides a research basis for the function study of PAP protein and the development of protein detection products for HLB.