Study On The Distribution Of Outer Membrane Protein W In Acinetobacter Baumannii Clinical Isolates And Its Pathogenicity

Objective:To analyze the different expression of outer membrane protein W in clinical isolates of Acinetobacter baumannii,and to research pathogenic features in vitro by cloning and expressing recombinant protein OmpW,and to provide new experimental datas and ideas for further clarifing drug resistance mechanism and looking for anti-infective targets.Methods:1.Anti-sera was obtained after immunization of animals with synthesized polypeptide.Anti-Omp W polyclonal antibody was obtained and purified after identified. 2. 31 Carbapenems resistant isolates(resistant group) and 30 susceptible isolates(sensitive group)were selected from clinical isolates of Acinetobacter baumannii.Expression of OmpW in two groups was detected by Western Blot.3.PCR method was used to amplify gene of ompW and ompA in Acinetobacter baumannii strain A1 and ATCC19606,and the prokaryotic expression vector was constructed. Recombinant protein OmpW and OmpA were expressed and purified after confirmed by sequencing.4.Human laryngeal epidermoid carcinoma cells were cultured with recombinant protein OmpW and OmpA, and the apoptosis of Hep-2 cells was detectived by JC-1 and DAPI. Results:1.The blue vector coupled with synthesized polypeptide vector was constructed successfully.Anti-Omp W polyclonal antibody was obtained from anti-sera after verified by mycoprotein abstracted from clinical isolates of Acinetobacter baumannii. 2.The positive expression rate of OmpW in Acinetobacter baumannii was 100% in resistant group and 63.33% in sensitive group by Western Blot. The expression of OmpW has a significant difference between two groups.The difference was statistically significant by Fisher exact probability( ? 2=12.359, P=0.000<0.01).3.The prokaryotic expression vector was successfully constructed. The gene was highly expressed in prokaryotic expression system(pET30a/ompW ? pET30a/ompA) after identified by gene sequencing. The gene molecular weight was 987 bp and 966 bp which was the same as expected. Deal with urea and Tris-HCl, the recombinant protein OmpW(42kD) and OmpA(41kD) were obtained and highly purified. 4. After outer membrane protein OmpW and OmpA affected on Hep-2 cells in apoptosis experiments, green fluorescence increased significantly by JC-1, which prompted the two proteins could induce apoptosis of Hep-2 cellsand blue fluorescence increased significantly by DAPI, which prompted the two proteins could lead to apoptosis of Hep-2 cells. Conclusions:1.Expression of OmpW in Acinetobacter baumannii resistant to Carbapenems was significantly higher than the sensitive strains.2.Outer membrane protein W could induce apoptosis of Hep-2 cells, which prompted that OmpW took part in multi-drug resistant Acinetobacter baumannii pathogenesis by particular route,but the specific mechanism need further study.