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| Cloning and prokaryotic expression of pilus formation protein gene from Candidatus Liberibacter asiaticus and preparation of its antiserum |
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| DOI：10.16768/j.issn.1004-874X.2018.11.012 |
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| Abstract: |
| 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 of 408 gene was obtained by PCR amplification with specific primers and sequence analysis showed that the 408 gene is identical with the homologue gene of the Candidatus Liberibacter asiaticus psy62 isolate (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, Motif 1 and Motif 2. The recombinant vector pET32a-408 was constructed by using EcoRⅤand XhoⅠ restriction enzyme sites. The pET32a-408 vector was transformed into E. coli BL21(DE3) and recombinant strain was induced by a final concentration of 1 mmoL/L IPTG. The 408 fusion protein was mainly expressed in the form of inclusion bodies. The 408 fusion protein was purified by Ni2+-NTA column and used to immunize mice by intraperitoneal injection. The 408 polyclonal antiserum was obtained and the titer was 1:500 ~ 1:10000. Western blotting further analysis showed that 408 polyclonal antiserum was specific to 408 protein. This study provides a research basis for the function study of 408 protein and the development of protein detection products for HLB. |
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