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Establishment Of The Novel Expression Systems In Mycobacteria And Their Application For Recombinant BCG

Posted on:2009-05-31Degree:DoctorType:Dissertation
Country:ChinaCandidate:X Y FanFull Text:PDF
GTID:1114360272488920Subject:Microbiology
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Tuberculosis(TB),though curable,still remains a major public health challenge worldwide.Mycobacterium tuberculosis(M.tb),the primary etiologic agent of TB, caused approximately 8 million new cases and 2 million deaths every year around the world.It has been estimated that 250,000 deaths occurred annually in China,among 6 million active TB patients at present.Furthermore,control of TB has been further complicated by the emergence of multidrug-resistant(MDR) M.tb strains and the human immunodeficiency virus(HIV) epidemic.The current vaccine,Mycobacterium bovis bacille Calmette-Guérin(BCG),a live attenuated vaccine derived from the bovine tuberculosis bacillus in the early 1900s,is still being administered to children in many countries,but its efficacy in protecting against TB,especially against the adult form of TB,remains controversial.BCG clinical trials showed its efficacy is highly variable,ranging from 0%to 80%.Thus,a new vaccine against TB more potent than BCG is urgently needed.Despite these unsatisfactory problems of BCG vaccine,its excellent immunostimulatory properties and proven safety for human use have led to the concept of recombinant BCG(rBCG)-based vaccines against TB and other infections. It has been reported that various protective candidate antigens could be cloned and expressed in BCG to make it more potent vaccine,by utilizing the E.colimycobacteria shuttle expression vectors.However,its application is restricted by slow-growing of BCG and low-level expression of recombinant plasmids.In the near past years,with the development of molecular biology and genetic engineering,there are many important progresses on rBCG expressing heterlogous immunodominant antigen,which shows a good prospect for its application.This study mainly focuses on establishing the mycobacterial high-level expression systems and to achieve the over-expression of target gene in mycobacteria.Based on the novel expression systems,the recombinant BCG vaccine strains with high-level expression of M.tb chimeric antigen,which showed excellent immunogenicity in our previous studies, were tried to construct,and were evaluated their immunogenicities in the animal experiments by analysis of the antigen-specific cellular and humoral immune response induced on the BALB/c mice.Using the regulatory region of M.smegmatis acetamidase(pACE) as promoter, the mycobacterial inducible expression vectors,pMF series,were constructed successfully.The acetamidase promoter was confirmed to be regulated tightly on the protein level,and the M.tb chimeric antigen was achieved to high-level expression in M.smegmatis by virture of the pMF series as expression vectors.The recombinant proteins obtained from M.smegmatis were mostly solube,which will be superiority over the same protein purified from E.coli expression systems.Furthermore,the addition of 6×His tag to the C-terminus of the recombinant protein facilitates the simple and effecitive purification of our model protein by Ni2~+-NTA affinity chromatography.However,the M.tb chimeric antigen was not expressed at high levels under the control of pACE in rBCG strains in spite of the addition of inducer acetamide;this suggests that the expression vectors based on the M.smegmatis acetamidase promoter seem not to be suited to drive expression of target antigen in BCG host.A simple and efficient promoter trapping vector,pMC210,was constructed with the promoterless E.coli lacZ gene as the reporter,modified on the E.colimycobacteiral shuttle plasmid pMV261.The M.tb furA gene promoter(pfurA) and its mutants were obtained by direct mutagenesis of the furA operator sequence,and these resulting mutated and the prototype pfurAs were then gene-fused to the immediately upstream of the lacZ reporter to analyze their in vivo transcrioption activities in M. smegmatis or M.bovis BCG.Theβ-galactosidase assay results showed that in both hosts,change of the initial codon GTG to ATG(pfurAα) only caused about twice increase of the transcription activity,while the 6-bp substitution in the 23-bp AT-rich region(pfurAm),which is conserved in the mycobacteria and is essential for FurA binding,caused about 4~6 times increase of the activity.With the combination of both mutations(pfurAma),theβ-galactosidase activity increased about 10 times,which was 1.7~2 times higher than the BCG phsp60,the strong promoter used widely for over-expressing proteins in mycobacteria.It is interesting to notice that the pfurA series exhibited higherβ-galactosidase activity in the slow-growing M.bovis BCG than in the fast-growing M.smegmatis.Subsequently,rBCG::lacZ strains transformed with the different pfurAs-lacZ construct were infected the murine macrophage RAW264.7 monolayer,the results showed thatβ-galactosidase activities were upregulated immediately in all of the rBCG::lacZ strains at the early infection,and were then persisted at levels higher than those recorded for rBCG::lacZ before macrophage infection,up to 7 days after infection.The next results on the animal experiment showed that all of mice immunized with the different rBCG::lacZ vaccines except pfurA-lacZ construct,were induced an enhanced Th1 immune response,characterized by increased production of antibodies of IgG2a subtype and higher secretion of IFN-γ.In addition,the amounts of T lymphocytes secreting IFN-γwere associated with theβ-gal levels expressed in the rBCG::lacZ strains.These suggests that pfurA series were seem to be suitable especially to drive gene expression in mycobacteria and rBCG vaccines based on pfurA series would induce production of antigen-specific Th1 immune response.Therefore,the prototype and the mutated pfurAs were used to construct mycobacterial differential expression vectors pMFA series.Employing the pMFA vectors,the M.tb chimericαg856α2 gene was expressed at various levels under the control of the different pfurAs in the recombinant M.smegrnatis and rBCG strains,and the double mutation on both 6-bp substitution in the FurA binding AT-rich region and GTG initiation codon to ATG variation,pfurAma,caused the highest level of gene expression level,which is consistent with the result ofβ-galactosidase assay. Subsequently,the BALB/c mice were immunized with the chimeric gene recombinant BCG(rBCG856A2) vaccines transformed with the different pfurA-αg856α2 constructs,and the results confirmed the good immunogenicities of rBCG856A2 vaccines,based on the anti-Ag85A and anti-ESAT-6 specific cellular and humoral immune responses.In order to facilitate the immune detection of rBCG856A2 vaccines,recombinant chimeric antigen Ag856A2(rAg856A2),rAg85A,and rESAT-6 were expressed and purified from E.eoli,respectively;and the murine anti-Ag85A and anti-ESAT-6 monoantibodies(mAbs) were also prepared.Next,we will focus on the challegene experiment on animal models in order to evaluate the immune protective efficacy,which would pave the way for the later clinical experiment of rBCG856A2 vaccines.
Keywords/Search Tags:Mycobacterium tuberculosis, Expression system, Promoter, Acetamidase, FurA, Chimeric gene, Recombinant BCG (rBCG)
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