Recombination,Expression And Biological Characterization Of BPSS1395 Protein Of Type ? Secretion System Of Burkholderia Pseudomallei

ObjectiveBurkholderia pseudomallei also was known as a kind of soil bacterium,which can invade into host cells.It leads to disease classfied as melioidosis.In addit ion,its infection can happen again in the latent years,so the melioidosis has been described as �Vietnamese time bomb�.Bacterial secretion systems have become the research focus on pathogenicity.Type ? secretion system(T3SS)plays an important role in bacterial virulence.As a putative gene,BPSS1395 is located in B.pseudomallei type ? secretion system.BLAST revealed that BPSS1395 has high sequence homology with HrpF protein(hypersentive response and pathogenicity F),which is an important transcription factor of Ralstonia solanacearum,belonging to the T3 SS effective protein.It is likely to transport and directional element of T3 SS.Therefore,we want to recombine biological BPSS1395 protein by genetic engineering and determine its subcellular localization,and produce the polyclonal antibodies against r BPSS1395.Then the function of BPSS1395 gene in the plant infection of B.pseudomallei was confirmed by knock out strains infecting tomato test.The study will lay the foundation for the further study of B.pseudomallei as a potential plant pathogens on human infection and transmisson.Methods1.The B PSS1395 gene were amplified by PCR with genomic DNA template of B.pseudomallei.The p ET-22 b plasmids carrying with BPSS1395 were transformed into E.coli BL21(DE3)cells.The positive clones were identified by ampicillin culture selecting,plasmid restriction enzyme digestion and DNA sequencing.2.The recombinant BPSS1395 prote in was expressed by IPTG inducing and purified by denaturation and recovery.3.The purified BPSS1395 protein was used to immunize rabbit,and its immunogenicity was identified by ELISA,Western blot and immunofluorescence test.4.Subcellular localization of protein BPSS1395 in B.pseudomallei was analysed by Western blot.5.Construction of BPSS1395 gene knockout strain: The upstream and downstream homologous arms of BPSS1395 gene were amplified,then connected to the pK18 mob Sac B suicide plasmid.The recombinant plasmids were introduced into BPC006 by conjugation with E.coli S17-1?pir.Target gene knockout mutation strain was selected by sucrose screening and identified by PCR and sequencing.6.Tomato roots were infected with BPSS1395 knockout strains,wild strains and EHEC O157:H7 seperately.Tomato leaves growth and morphology were observed,and bacterial colonization was detected by PCRResults1.The BPSS1395 gene was obtained by PCR from the genomic DNA of B.pseudomallei and was successfully inserted into expression plasmid p ET-22 b.And the recombinant bacteria p ET-22b-BPSS1395/ BL21(DE3)were constructed2.The protein with MW 32 kDa was expressed and prepared.3.The rabbit polyclonal antibodies against rBPSS1395 were produced,with 1:1280000 ELISA titer.4.It was shown that BPSS1395 mainly localized in the cytoplasm of B.pseudomallei.5.BPSS1395 gene knockout strains of B.pseudomallei were constructed by homologous recombination.6.No obvious morphology changes were observed in tomato roots and leaves infected between BPSS1395 knockout strains and wild strains,but the coloniz ation of B.pseudomallei was existed in wild strains infected group by electron microscope and PCR,no colonization in knockout strains group.ConclusionsBPSS1395 protein with biological activity was recombinated,and its subcellular localization was determined.The polyclonal antibody against r BPSS1395 provides an effective tool for the study of bacteria and protein function.It was confirmed that BPSS1395 plays an important role in plant infection with B.pseudomallei.This study provides effective suggestion for the prevention and control of B.pseudomallei infection.