Expression And Nucleic Acid Immune Of 47 KDa Protein Gene Of Orientia Tsutsugamushi Strain Karp

Background Tsutsugamushi disease is a kind of zoonotic infection caused by Orientia tsutsugamushi, which spreads wildly in our country's southern command and southeast Asia as well as the Pacific region. During World War II, the disease had taken more victims than the casualties due to the war itself in Southeast Asia and the southwestern Pacific region. The epidemic disease is still go forward in the northern parts of our country even now.The typical symptom of tsutsugamushi disease is high fever, eschar, lymphadenopathy and may progress to disseminated intravascular coagulation, multiorgan function failure, or even death. A quarter of the patients were children. It is still very difficult to provent the disease because of the lack of vaccine for Orientia tsutsugamushi.Orientia tsutsugamushi is a kind of intraceller bacteria which can hardly be killed by host's antibody. The cell-mediated immunity induced by its antigen secreted in host cell is the host's important protecting machiniam.Nucleic acid vaccine in host's cell can induce humoral immunity with MHC II and cell-mediated immunity with MHC I at the same time, which may be effective for the interceller bacteria.47kDa protein is the mainly protective antigen on the membrane of Orientia tsutsugamushi with group-specific epitopes and strain-specific epitopes for B cell and T cell on it. The gene may be study as one of the important genes for nucleic acid vaccine.Objective To amplify and clone the 47kDa protein gene that code the whole mat peptide of Orientia tsutsugamushi Karp strain by PCR . To construct the E. coli expression plasmid and express 47kDa protein in vitro, then test its immunogenicity and antigenicity. To construct mammalian cell expression plasmid and study the nucleic acid immunogenity of the 47kDa protein gene.Methods(1) Two pairs of primers were disigned according to the published 47kDa protein gene sequence of Orientia tsutsugamushi Karp strain, one for the construction of prokaryotic expression plasmid and the other for that of eukaryotic expession plasmid. The whole sequence of mat peptide of the 47kDa protein gene was amplified and cloned to T vector by TA clonetechnic to construct plasmid pMD18-T-47 or pMD18-T-47ISS which were identified by restriction analysis and PCR.(2)Then the mat peptide gene was subcloned into a E.coli expression vector to construct a recombinant plasmid pGEX-47. The E.coli cells DL21were transformed with the recombinant plasmid pGEX-47 and transformers were induced to express the recombinant protein by IPTG. The recombinant 47kDa protein expressed by pGEX-47 was identified by electrophoresis as well as its immunity characteristics by western blot analysis and indirect immunofluorescent antibody assay (IFA) of sera of the immunized mice.(3)The 47kDa protein gene amplified with CpG DNA as a vaccine adjuvant was subcloned to a mammalian cell expression plasmid pVAXl to construct the recombinant plasmid pVAX1-47. The recombinant plasmid pVAX1-47 was transfected into Hela cell by Lipofectamine to achieve the expression in vitro and the antigenicity of the expression productwas tested by western blot assay and indirect immunofluorescent antibody assay. After verifying that the 47kDa protein could be expressed in mammalian cells and that it could immunize murine as described above. the recombinant plasmid pVAX1-47 were administered in BALB/c mice to study its immunogenicity by testing the antibody titers in sera and the proliferation of T cell of the immnized mice.Result(1) The PCR product of 47kDa protein gene of Orientia tsutsugamushi Karp strain was obtained and its sequence was the same as the published 47kDa protein gene.(2) The E.coli expression plasmid pGEX-47 constructed was identified containing the 47kDa protein gene fragment by restrication analysis and PCR identification. The recombinant plasmid pGEX-47 expressed GST-47 fusion protein in prokaryotic cell by IPTG. The 47kDa protein purofied from GST-47 fusson protein induced a high antibody(IgG) titers of 1:1280 in sera o...