Prokaryotic Expression Of Secreted Proteins S9 And S10 From Candidatus Liberibacter Asiaticus And Preparation Of Their Antisera

Citrus Huanglongbing?HLB?caused by Candidatus Liberibacter spp.is the most destructive disease of citrus worldwide,and it is widely distributed in Asia,Africa and the Americas.In China,more than 300 counties from 11 provinces have been suffering damage from HLB.So far,there is no curable drugs available for HLB.Its most aggressive pathogen is C.Liberibacter asiaticus?CLas?,a gram-negative and phloem-restricted bacteria,which is mainly transmitted by Diaphorina citri in the field.Since the pure cultivation of CLas in vitro is still of most difficulty,the in-depth study of its physical and chemical characteristics and the development of disease management technologies are facing great challenges.The complete genome sequence of CLas was published in 2009,which showed that it has a Sec-dependent pathway and secretory function.Secreted proteins play a crucial role for the interaction of plant-pathogen,but the research on interactions of its secreted proteins and citrus host remains unclear.It is a common method to prepare antibodies for exploring the interaction between pathogen and host plant.In this study,two secreted protein genes of CLas were screened,and antisera were prepared with their expressed proteins in vitro,which laid a preliminary foundation for studying the functions of these two secreted protein genes,and also provided a reference for establishing serological detection method and screening method of secreted protein genes of HLB.The main results were as follows:1.Screening of secreted protein genes from CLasBased on the 166 predicted secreted protein genes of Prasad et al.,three-point screening conditions were addressed: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 have secretory function which were verified by alkaline phosphatase?PhoA?;3)the protein size is between 10 kDa and 25 kDa.Seven secreted protein genes were preliminary screened.Reverse transcription-polymerase chain reaction?RT-PCR?was used to determine the expression of potential CLas secreted proteins in infected citrus,only secreted protein genes S9?CLIBASIA?05150?and S10?CLIBASIA?04040?were amplified.Blast analysis using the nucleic acid sequences of S9 and S10 from the genome of CLas-psy62.The S9 is highly conserved among the genome sequences of six CLas strains with 100%identity in nucleotide.The S10 has 100%identity with CLas-A4,and there is one base difference in nt of the other four CLas strains.Amino acid sequence alignment revealed a difference at the 135^thh amino acid.There were five potential domains predicted by InterPro,four of which didn?t contain the different site.Therefore,the secreted proteins S9 and S10 were chosen for further study.2.Prokaryotic expression and protein purification of secreted proteins S9 and S10Design specific primers based on S9 and S10 nucleic acid sequences of CLas-psy62.We amplified the secreted protein genes screened from infected samples collected in October from Jiangxi province by polymerase chain reaction?PCR?.The expression vectors pET-28a-S9 and pET-28a-S10 were successfully constructed for the secreted protein genes based on vector pET-28a,and the expression vectors were transferred into Escherichia coli DH5?.Positive clones were verified by double digestion and sequencing.The cloned sequences were 100%identity with the S9 and S10 sequences of CLas-psy62,respectively,and the coding frames were in the correct direction without shifting.The recombinant expression vectors pET-28a-S9 and pET-28a-S10 were transformed into E.coli BL21?DE3?successfully.The expression strains were induced to express by the temperature and IPTG concentration,Western blot were addressed to verify that two target proteins were expressed successfully.The optimized induction temperature and isopropyl-beta-D-thiogalactopyranoside?IPTG?concentration of two expression strains were 18?and 0.1 mM,respectively.The fusion protein S9 was insoluble in the mixed solution instead of the supernatant,indicating the form of inclusion bodies.The S10 was soluble in both the mixed solution and the supernatant,indicating S10 is a soluble protein.The fusion protein S10 was purified by Ni²⁺-NTA affinity chromatography.It was better in phosphate buffer at pH 8.0 and mainly in the 30-50 mM elution buffer without other proteins.Western blot analysis showed that it was the target protein S10.3.Preparation of antisera and their potency analysisEach of purified fusion proteins was approached as antigen to immunize rabbits intraperitoneally to obtain S9 and S10 antisera.Then direct binding of the antibody with the purified fusion protein was evaluated using indirect enzyme-linked immunosorbent assay?ELISA?,the titer of S9 antisera for indirect ELISA was 1:4000 and the sensitivity1:32000.Dot ELISA was addressed to assure its color reaction with infected samples from field,the positive dot ELISA detections accounted for 38.9%of the positive PCR detections,of which samples were mainly collected in September.Western blot was approached to assure that S9 antisera could hybridize with the fusion protein but not with the infected citrus.The titer of S10 antisera for indirect ELISA was 1:4000 and the sensitivity 1:512000.Dot ELISA was used to assure its color reaction with infected samples from field,the positive dot ELISA detections accounted for 33.3%of the positive PCR detections,of which all samples were mainly collected in September.Western blot was used to assure that the S10 antisera could only hybridize with the fusion protein.It is suggested that the secreted proteins S9 and S10 are related to CLas,but more field samples are still needed to be tested for optimizing the conditions.They can also be used to establish effective detection methods.