中国生物工程杂志

Objective:To construct shuttle expressing plasmids of H₃₇Rv eis gene, and identify biological activity of recombinants in Mycobacterium smegmatis mc²155(M.S).Methods: eis gene was amplified from Mycobacterium tuberculosis genomic DNA and clonded into an Escherichia coli-Mycobacteria shuttle plasmid pMV261 to construct a recombinant plasmid pMV-eis. The pMV-eis was performed successfully by PCR, enzyme digestion analysis and sequencing. pMV-eis then was transformed into M.S by electroporation. After cultivation positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again.The expression of Eis protein in the M.S was proved by SDS-PAGE and Western blot. Results: Sequencing and homologous analysis veryfied the cloned eis gene, and its nucleotide and deduced aminoacid sequences was equaled to the related gene in GenBank.The growth curve of the recombinant Mycobacterium smegmatis(rM.S) was consistent with that of the wild-type strain, suggesting the absence of Eis protein toxicity against M.S.SDS-PAGE and Western blot result showed that relative molecular mass of expression product was about 42kDa. Conclusion:The shuttle plasmids pMV-eis has been constructed successfully. and the recombinant plasmid has biological activity in rM.S, which lays the foundation for further research about of Eis function.