Recombinant Expression And Biological Characterization Of Burkholderia Pseudomallei Toxin BLF1

Burkholderia pseudomallei,a gram-negative pathogen,which extensively exists in soil and water,is the causative agent of melioidosis.The classical characteristic of the disease is pneumonia,pulmonary cavity and multiple abscesses.Melioidosis is endemic in northern Australia,southeast Asia and other tropical regions.In Thailand,melioidosis is the most common cause of pneumonia-derived sepsis.Recent researchs show that endemic zone has also expanded to areas of Pacific and Indian Ocean islands,and parts of the Americas.With a mortality rate up to 40%,B.pseudomallei has been classified as category B potential bioterrorism agent by the Center for Disease Control.The numerous features of melioidosis includ long latency,recrudescence and antibiotic resistance.A human vaccine is currently no available for melioidosis,which indicates that increasing research and understanding of melioidosis is imminent.Virulence factors play an important role in pathogenesis of B.pseudomallei.Multiple potential virulence factors have been discovered for B.pseudomallei,such as type III secretion system,type VI secretion systems,capsular polysaccharide,lipopolysaccharide,flagella,the quorum-sensing system,fimbriae and so on.BLF1(Burkholderia lethal factor 1)coded by bpsl1549 is structurally similar to the C-terminal domain of the Escherichia coli cytotoxic necrotizing factor 1 and mainly inhibits helicase activity of translation factor e IF4 A,finally the protein synthesis is inhibited and death of cells occurs.The discovery of BLF1 in B.pseudomallei provides a new thinking in pathogenic mechanism and plays an important role on diagnosis,therapy and prevention of melioidosis.Recent years,more and more inhibitors of protein synthesis have been described as anticancer agents,including inhibitors of eIF4 A.BLF1 is a potential eIF4 A helicase activity inhibitor,we have a hypothesis that if deliveried to tumor cells,does BLF1 have the effect of broad-spectrum killing tumor cells? There is no research focusing on the interaction of BLF1 and tumor.This study is to recombine and express B.pseudomallei toxin BLF1 and preliminarily study its cell-killing effect,providing evidence for the application of the protein in anticancer therapy.Methods1.The bpsl1549 gene was amplified by PCR with the genomic DNA of BPC006 strain.The double enzyme digested products of bpsl1549 and p GEX-6P-2 were transformed into E.coli XL1-Blue cells,culturing on LB solid medium with ampicillin.Positive clones were identified by restriction enzyme digestion and DNA sequencing.2.Fusion protein GST-BLF1 was induced by IPTG and purified by affinity chromatography.The immunogenicity of the protein BLF1 was examined by ELISA and Western blot.3.The purified protein was acted on A549 cells to study its cytotoxicity and the blocking effect of antibody by detecting the number of living cells through the CCK-8 kit.Observe the survival rates and viscera pathological changes after intraperitoneally injecting protein r BLF1 of different density in BALB/c mice.Results1.The bpsl1549 gene was obtained successfully by PCR amplification and the recombinant plasmid that highly expressed fusion protein GST-BLF1 was constructed successfully.2.The expressed protein r BLF1 was about 23 k D.The titre of the multiclonal antibody reached to 1 280 000;a specifical band was found in the target protein position by Western blot.3.Protein rBLF1 obviously killed A549 cells and antibody could inhibit its cytotoxicity effectively.4.After intraperitoneal injection of r BLF1 protein,the mortality rate of BALB/c mices positively correlated with the dosages of protein and there are no significal pathological changes between control group and experimental group.ConclusionrBLF1 with biological activity was sucessfully expressed,the protein had obvious effect to kill A549 cells and was toxic to normol cells,but normol cells tolerated the toxin to some extent.