| BackgroundBacilli pseudomallei is called Burkholderia pseudomallei academically, which wasfirstly indentified as pathogen in Yangon of Myanmar by Whitmori and Krishnaswami in1912. Because of its morphological and cultural characteristic similarities and obviousserological cross-reactivities to Burkholderia mallei, it was named Burkholderiapseudomallei formally in1993and was then accepted world wide. Burkholderiapseudomallei is an important zoonotic pathogens, and the outbreak of which at differentscales were reported all over the world besides China. The Burkholderia pseudomalleimainly grow in soil and water, which infected people by the respiratory tract, digestive tract,bites of blood-sucking insect and other ways.Be similar to the manifestations of Mycobacterium tuberculosis infection, the clinicalsymptoms of Burkholderia pseudomallei infectious diseases used to be as the following:multiple organ infections, refractory pneumonia, lung cavities, sepsis and even death.They infection are classified into acute sepsis, sub-acute infection, chronic infection andsubclinical infection base on the pace of diseases progression. Acute sepsis type is themost serious type with a mortality of up to60%. The current mortality is still higher than30%despite of the development of antibiotics. The infection Burkholderia pseudomalleihas three characteristics containing a trends of outbreak, strong pathogenicities andlethalities and exacerbated condition by antibiotic treatment, which make it become aglobal public health problem. However, their is still a lack of effective strategies fordiagnosis, prevention and treatment. Due to the difficulties in control of Burkholderiapseudomallei, to develope vaccines becomes simple, economic and effective way tostruggle with Burkholderia pseudomallei.Flagellin (flagellin, FliC), a main bacterial surface antigen, is able to elicit effectiveimmune response. Meanwhile its amino acid sequence is highly conserved. As a result, it has been widely used for the diagnosis of bacterial infections and becomes a subunitvaccine candidate. we firstly tried to express the recombinant FliC in pET22b, pET28aand pGEX but failed. In our previous study, we identified EspA, a component of the type IIIsecretion system from EHEC O157: H7, which is able to promote the expression ofexogenous proteins. In addition, we constructed pEspA plasmid for the expression ofEspA fusing proteins. In this study, we plan to prepare recombinant FliC with the help ofpEspA plasmid. Moreover, the immunological properties of EspA-FliC will be evaluatedfor the future development of bivalent vaccine against Burkholderia pseudomallei.Methods1Construction of recombinant plasmidsGenomic DNA extracted from Burkholderia pseudomallei was used as the PCRtemplate. Gene bpsl3319encoding FliC was amplified and cloned into pET22b, pET28a,pGEX and pEspA separately. The recombinant plasmids were verified by double digestionand DNA sequencing.2Expression and purification of recombinant proteinsThe plasmids were transformed into Escherichia coli BL21and induced by IPTG. Thewhole cells were collected and analysised by SDS-PAGE. The recombinant EspA-FliC waspurified by anion exchange chromatography and gel filtration chromatography. Theconcentration of EspA-FliC was detected by BCA method.3Immunological properties of EspA-FliC3.1Immunological reactivity of EspA-FliC. Western blot was used to detect therelativities of EspA-FliC to anti-Burkholderia pseudomallei polyclonal antibodies,anti-EspA polyclonal antibodies anti-EspA monoclonal antibody.3.2Immunogenicity of EspA-FliC. Purified EspA-FliC was used to immunize Balb/cmice. EspA-FliC specific antibodies were detected by ELISA. In addition, Western blot wasused to detect the binding of immunized serum to recombinant EspA, EspA-FliC and nativeFliC from Burkholderia pseudomallei.Result1The recombinant plasmid pET22b-bpsl3319、pET28a-bpsl3319、 pGEX-bpsl3319and pEspA-bpsl3319confirmed by enzyme digestion assay and DNA sequencing. Theexpression of FliC was only found in pEspA expression system. 2EspA-FliC accounted for about35%of total protein after induction of IPTG. Thepurity of recombinant EspA-FliC reaches as high as85%after careful purification.2Results from western blot showed EspA-FliC was able to interact with anti-Burkholderia pseudomalle polyclonal antibodies, anti-EspA monoclonal antibody andanti-EspA polyclonal antibodies simultaneously.4Specific IgG was detected in EspA-FliC immunized mice, which was able torecognize recombinant EspA, EspA-FliC and native FliC from Burkholderia pseudomalle.These results suggested the good immunogenicity of EspA-FliC.ConclusionIn this study, EspA from enterohemorrhagic Escherichia coli O157: H7and FliC fromBurkholderia pseudomallei was successfully fused and expressed by recombinant DNAtechnologies. In addition, it showed remarkable immunogenicity and immune reactivity.Our results laid solid foundations for the future development of combined bivalent vaccineagainst Burkholderia pseudomallei and enterohemorrhagic Escherichia coli O157: H7. |
PDF Full Text Request