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| Prokaryotic Expression and Purification of OmpA of Klebsiella pneumoniae Isolated from Mauremys mutica |
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| DOI：10.16768/j.issn.1004-874X.2023.03.016 |
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| Abstract: |
| 【Objective】The study was conducted to clone the outer membrane protein A (KpOmpA) gene of Klebsiella pneumoniae isolated from Mauremys mutica, construct the recombinant plasmid and induce its expression, purify and obtain the recombinant protein KpOmpA, which would lay a foundation for preparing the vaccine to prevent K. pneumoniae infection. 【Method】Using the DNA as the template, KpOmpA gene was cloned and connected with pET-32a expression vector, and the constructed recombinant plasmid pET-32a-KpOmpA was transformed into the competent cells of E. coli BL21 (DE3). After induction by IPTG, the expression of recombinant protein KpOmpA was detected by using SDS-PAGE and the dissolubility was analyzed. Then the KpOmpA was purified by Ni-IDA agarose resin column. In addition, purified recombinant protein was identified by Western blot.【Result】The CDS sequence of KpOmpA was 1 071 bp in length with presumptive encoding 356 amino acids. The OmpA was highly conserved which shared more than 99% sequence homology with those from other K. pneumoniae strains. The pET-32a-KpOmpA was identified and confirmed by double enzyme digestion and sequencing. The sequencing result confirmed that there was no code shift or mutation, indicating that the recombinant plasmid was successfully constructed. The transformed competent cells of E.coli BL21 were induced by IPTG with the final concentration of 1 mmol/L at 37 ℃ for 4 h, SDS-PAGE assay showed that KpOmpA was expressed abundantly in the form of inclusion bodies, with a molecular weight of about 57 kD. The recombinant protein was purified and obtained by Ni-IDA agarose resin column. Western blot results show that there was a 57 kD band, indicating that recombinant protein KpOmpA could be recognized by His-tag mouse monoclonal antibody.【Conclusion】The recombinant plasmid pET-32a-KpOmpA was successfully constructed and expressed in E. coli expression system. The recombinant protein KpOmpA with high purity was obtained, laying a foundation for preparing the vaccine to prevent K. pneumoniae infection. |
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