An Investigation on the Molecular Characteristics and Intracellular Growth Ability among Environmental and Clinical Isolates of Legionella pneumophila in Sichuan Province, China.

OBJECTIVE: To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila (L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. METHODS: Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing (SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci (MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. RESULTS: All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types (STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtxA loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. CONCLUSION: L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.

[Preparation of recombinant Mycobacterium tuberculosis ESAT6-PPE68 fusion protein].

OBJECTIVE: To construct esat6-ppe68 fusion gene and its prokaryotic expression vector for expression in E. coli. METHODS: With GeneSOEing method, a fusion gene was constructed by splicing esat6 gene and ppe68 gene and cloned into pGEX-4T-1 plasmid to construct the recombinant prokaryotic expression plasmid pGesat6-ppe68. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis of the plasmid, E. coli BL21 was transformed with the recombinant plasmid and induced with IPTG to obtain the expression of the fusion protein ESAT6-PPE68 with GST-tag (about 69 kD), which were purified with GST-fusion protein purification kit. The expression of esat6-ppe68 fusion gene was subsequently detected by SDS-PAGE and Western blot analysis. RESULTS: The sequence of esat6 and ppe68 in the recombinant plasmid was consistent with that in GenBank report. The fusion protein was detected in the cytoplasm in soluble form and represented approximately 40% of the total bacterial protein of E. coli. After purification, the purity of the fusion protein reached 90%, and its antigenicity was confirmed by Western blotting. CONCLUSION: The prokaryotic expression vector pGesat6-ppe68 has been constructed and the fusion protein ESAT6-PPE68 obtained successfully, which provides an experimental basis for potential application of the recombinant ESAT6-PPE68 in the diagnosis of tuberculosis.