Screening and prokaryotic expression of secreted protein 05150 from Candidatus Liberibacter asiaticus and preparation of its antiserum

Abstract:【Objective】Citrus Huanglongbing (HLB) is the most destructive disease of citrus world- wide. In China, HLB is caused by Candidatus Liberibacter asiaticus (CLas), a gram-negative, phloem- restricted and psyllid-transmitted bacterium. The citrus industry is facing an unprecedented challenge. At present, the world’s difficult problem of pure cultivation of CLas has not been overcome. The in- depth study on its physical and chemical characteristics and the development of disease management technologies are facing great challenges, and the research on the interaction between secreted proteins and citrus is not clear completely. In order to research the function of secreted proteins of CLas, one secreted protein was screened, expressed in vitro, and its polyclonal antibodies were made.【Methods】Based on the 166 predicted secreted protein genes of Prasad et al., four-point screening conditions were set: 1) the genes can be analyzed by using all the four softwares: Phbius, SigP4.1, LipoP, and SigP3.0; 2) the predicted signal peptide of genes has secretory function that has been verified by alkaline phos-phatase (PhoA); 3) the protein size is between 10 ku and 25 ku; 4) the expression of potential CLas se- creted proteins in infected citrus can be determined by reverse transcription-polymerase chain reaction (RT-PCR). We amplified the secreted protein gene screened from infected samples collected from Ji- angxi province by PCR. The expression vector with Nde I (F) and Xho I (R) restriction sites was suc- cessfully constructed for the screened secreted protein gene based on vector pET-28a, and the expres- sion vector was transferred into Escherichia coli BL21 (DE3). The expression strain was induced to ex- press by the IPTG concentrations, which were set to 0, 0.1, 0.3, 0.5, 0.7 and 1.0 mmol·L-1. Then the op- timal induction IPTG concentration was selected to induce the expressed protein, and Western blot was addressed to verify whether the target protein was expressed using Anti-his-tag mAb and Anti-IgG (H+ L chain) (Mouse) pAb-HRP. The purified fusion protein was approached as an antigen to immunize rab- bits intraperitoneally to prepare polyclonal antibodies. Then direct binding of the antibody with the puri- fied fusion protein was evaluated using indirect enzyme-linked immunosorbent assay (ELISA), ELISA plates were coated with 100 μL of the purified fusion protein (1:4 dilutions in carbonate coating buffer), the concentrations of the polyclonal antibodies were set to 1∶500, 1∶1 000, 1∶2 000 ...... 1∶8 192 000 and 1∶16 384 000, respectively, and Anti-IgG (H+L chain() Rabbit)pAb-HRP was diluted to 1∶8 000 with 1 × PBS buffer. Then dot ELISA and Western blot detection methods were addressed to verify the effectiveness of the polyclonal antibodies with the field samples. The concentrations of the polyclonal antibodies were set to 1∶1 000, 1∶3 000, 1∶5 000, 1∶7 000, and 1∶10 000, respectively. Anti-rabbit IgG (whole molecule)-AP or Anti-IgG (H+L chain) (Rabbit) pAb-HRP was diluted to 1∶8 000 with 1 × PBS buffer. Total protein was extracted by 1×PBS buffer from field samples collected in May, September, and November from Jiangxi, Sichuan, and Fujian provinces, respectively.【Results】In this study, six se- creted protein genes were screened preliminarily from the 166 predicted secreted protein genes, and in- direct verification of six secreted proteins expression was conducted in infected citrus from Jiangxi province by RT-PCR. We only amplified the 05150 sequence in infected citrus, which was highly con- served among six CLas strains whose genome sequences were available with 100% identity in nucleo- tide sequences. CLIBASIIA_05150 was screened, and the protein size was approximately 22 ku (i.e., excluding the predicted N-terminal signal peptide, 1-33 aa). The 05150 sequence was ligated to the vec- tor pET-28a, and the expression vector pET-28a-05150 was successfully constructed, and pET-28a- 05150 was transferred into E. coli BL21 (DE3) successfully. The optimized induction for final IPTG concentration of the expression strain pET-28a-05150 was 0.1 mmol·L-1, and fusion protein was insolu- ble in the mixed solution instead of the supernatant, indicating the formation of inclusion bodies. The pET-28a-05150 polyclonal antibody was evaluated for binding affinity to the pET-28a-05150 antigen by indirect ELISA. The binding affinity was tested using different concentrations of the polyclonal anti- body, confirming the optimal concentration of pET-28a-05150 polyclonal antibody for indirect ELISA 1∶4 000 and the sensitivity 1∶32 000. Dot ELISA assured its color reaction with infected samples from the field. The positive dot ELISA detections accounted for 38.9% of the positive PCR detections, of which 4 and 2 samples were collected from Jiangxi province in September and May, respectively, 1 from Sichuan province in September. Western blot assured that pET-28a-05150 polyclonal antibody hybridized with the fusion protein but not with infected citrus.【Conclusion】In this study, secreted protein gene 05150of CLas was screened, and pET-28a-05150 antiserum was prepared with its expressed protein in vitro, which laid a preliminary foundation for studying the function of 05150, and also provided a reference for establishing serological detection and screening methods for secreted protein gene of HLB.