Prokaryotic recombinant expression of Mycobacterium tuberculosis Lsr2 protein andits polyclonal antibody preparation
SUN Wei-guo1, HOU Jiang-hou2, LI Gai-ping3, YANG Li-kun1, SUN Wen-na1, ZHANG Ling-xia1
1. Army Tuberculosis Prevention and Control Key Laboratory, Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Institute for Tuberculosis Research, the Eighth Medical Center of General Hospital of PLA, Beijing 100091,China; 2. Maternal and Child Health Care Hospital of Kunming City, Kunming, Yunnan 650013, China; 3. Department of Respiratory Medicine, the 309th Hospital of Chinese PLA, Beijing 100091,China
Abstract:Objective To obtain recombinant Mycobacterium tuberculosis (M. tuberculosis) protein Lsr2 in prokaryotic expression system, and to prepare its polyclonal antibody. Methods Primers of Lrs2 sequence were synthesized according to the sequence of the M. tuberculosis H37Rv genome. Lrs2 was amplified by PCR and cloned into prokaryotic expression vector pET-28a. The recombinant plasmid pET-28a-Lrs2 was transformed into Escherichia coli Rosetta (DE3). The expression of the recombinant protein was induced with isopropyl β-D-thiogalactoside (IPTG), then renatured and purified by metal chelate chromatography. New Zealand rabbits were immunized to prepare polyclonal antibody against Lrs2. The antibody was identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting assay. Results pET-28a-Lrs2 was constructed successfully. Recombinant Lrs2 protein had a relative molecular mass of about 14 kD according to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After affinity purification and on-column refolding, the purity of the recombinant protein reached about 95%. The titer of the prepared polyclonal antibody was more than 1∶5.5×106, and the antibody could recognize the purified recombinant Lrs2 protein or Lrs2 protein in M. tuberculosis H37Rv lysate. Conclusion The recombinant M. tuberculosis protein Lrs2 is successfully expressed and purified in prokaryotic expression system, and anti-Lrs2 polyclonal antibody with high specificity is obtained, which lays a foundation for further studying the function of Lrs2, screening its interaction proteins and investigating its related molecular mechanisms.
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