Preparation And Antigenic Analysis Of Recombinant Outer Membrane Protein Pap31 Of Bartonella Henselae

【Background and Objectives】Bartonella henselae is a gram-negative bacterial pathogen of humans and animals. Infection of humans with B. henselae results in"cat scratch disease", its pathogenesis is unknown. It has been reported that the growth of B. henselae has high requirement of hemin, but B. henselae can't produce hemin itself. The outer membrane protein Pap31 is the mainly hemin-binding protein, by which B. henselae gets iron is necessary for growth. In addition, Pap31 is an extracellular adherin, it is involvd in recognizing and binding the host cell surface, which is the first step of bacterial colonization and invasion. All suggest that Pap31 is an important causative agent. The present study is to prepare recombinant outer membrane protein Pap31 of B. henselae and to analyze its antigenicity, and provide a theory basis for the development of diagnostic reagent and vaccine.【Methods】1. Amplification of pap31 gene by PCR: Primers for the amplification of the outer membrane protein Pap31 gene of B. henselae were designed according to the nucleotide sequence deposited in GenBank (accession number: AF001274), BamH I and Hind III sites were included in the forward and reverse primers. The B. henselae Pap31 protein gene was amplified by PCR.2. Construction of the recombinant plasmid pQE30/pap31: The pap31 gene fragment and the plasmid pQE30 were digested with BamH I/Hind III, then ligated them and transformed to competent E. coli M15. The recombinant plasmid pQE30/pap31 was confirmed by double enzymes cut and PCR.3. Expression of the recombinant protein Pap31:E.coli M15 transformed with the pQE30/pap31 plasmid DNA was induced by the addition of 1 mmol/L Isopropyl h-d-thiogalactopyranoside (IPTG). After the addition of IPTG, the recombinant bacterium was analysized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)4. Immunoreactivity analysis of Pap31 by Western Blot: Proteins were separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane, and then incubated with primary and second antibody in turn, observed specific binding bands after DAB coloration.5. Preparation of polyclonal antibody:Mice were immunized by recombination protein Pap31 with standard method, then detected sera from mice immunized with Pap31 by indirect immunofluorescence assay (IFA)【Results】1. Amplification of pap31 gene by PCR: Using B. henselae genomic DNA as the template,a 1000 bp gene fragment was amplified by PCR, which is the same size as expected.2. Construction of recombinant plasmid pQE30/pap31: Identification of the recombinant plasmid pQE30/pap31 by PCR and double enzymes cut, which shows the aim gene was inserted successfully in the vector pQE30.3. Expression of recombinant plasmid pQE30/pap31 in E.coli M15: Analysis of the induced E.coli M15 transformed with the pQE30/pap31 by SDS-PAGE, there is a protein band between molecular weight standard 25kD and 35kD, whose molecular weight is about 31 kD and is as same as expected.4. Analysis of Western-blot:DAB staining shows that pQE30/pap31-transformed E. coli M15 induced with IPTG has a 31kD immunoblotting band, but the band doesn't exist in pQE30/pap31-transformed E. coli M15 without induction; pQE30-transformed E. coli M15 induced with IPTG; and pQE30–transformed E. coli M15.5. Detection of sera from mice immunized with Pap31 by IFA:The whole cell antigen of B.hensaleae was recognized by the sera form mice immunized with the recombinant protein Pap31 in indirect immunofluorescence assay, and the titer of all the five mice is 1:5120. The same sera can't be recognized by the antigen of Bartonella quintana andCoxiella burnetii.【Conclusions】The recombinant plasmid pQE30/pap31 is constructed successfully. The pap31 gene of B. henselae is efficiently expressed in pQE30/pap31-transformed E. coli and the expressed recombinant protein Pap31 is an efficient antigen to induce antibodies against B. henselae and to react with B. henselae-immunized sera. Pap31 is one of potential candidate molecules for the development of diagnosis reagent and vaccine against the infection of B. henselae. 【Background and Objectives】Bartonella quintana is a reemerging, gram-negative, facultative intracellular bacterium which causes a variety of human diseases, including trench fever, bacteremia, endocarditis, and bacillary angiomatosis. Human is the only known host and the confirmed transmission vector is the human body louse. Very little is known about the pathogenicity of B. quintana. Heme is a source of iron for a variety of gram-negative bacteria, it has been reported the growth of B. quintana has highest requirement of hemin for a bacterium, but B. quintana can't produce hemin itself. The outer membrane protein HbpA is the dominant hemin-binding protein, by which B. quintana gets iron, and it is necessary for growth. In addition, recent work has shown that Fab fragments purified from a polyclonal antibody specific for HbpA were able to significantly decrease the amount of hemin bound by B. quintana in a dose-dependent manner. All those suggest that HbpA is an important causative agent. The present study is to prepare recombinant outer membrane protein HbpA of B. quintana and to analyze its antigenicity, and to provide a theory basis for the development of diagnostic reagent and vaccine.【Methods】According to the gene sequence of Gene Library, DNA primers were designed. The hbpA gene fragment was amplified from genomic DNA of B. quintana and cloned into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32a(+)-hbpA. E. coli cells were transformed with the recombinant plasmid and the transformants were induced to express the target protein by IPTG, and then the expressed recombinant protein was confirmed by SDS-PAGE and Western Blot assay.【Results】The recombination protein HbpA was expressed successfully in the prokaryotic expression system. In SDS- PAGE and immunoblot analysis, a 48 kDa recombinant protein was shown in E. coli cells transformed with the pET32a (+)-hbpA and it was recognized by sera from mice immunized with HbpA; the whole cell antigen of B. quintana was recognized by the sera from mice immunized with the recombinant protein in indirect immunofluorescence assay. Cross-reactivity between the B. quintana HbpA protein and the sera of mice immunized with B. henselae and Coxiella burnetii was not observed in the present study.【Conclusions】The hbpA gene of B. quintana is efficiently expressed in pET32a (+)-hbpA -transformed E. coli and the expressed recombinant protein HbpA is an efficient antigen to induce antibodies to B. quintana and to react with B. quintana -immunized sera. HbpA is a potential candidate molecule for the development of diagnosis reagent and vaccine.