Preparation And Identification Of Recombinant Ghost Vaccine Expressing HpaA Of Helicobacter Pylori

Helicobacter pylori (Hp) is a gram negative,spiral or S-like bacterium that resides in stomach, which may cause diseases in upper alimentary tract such as chronic gastritis, gastric ulcer, duodenumulcer. Because of many disadvantages with the traditional antibiotic treatment such as high cost, antibiotic resistance, it has become a hot topic to develop efficient vaccine to prevent or cure Hp infection. Most of currently developing Hp vaccines contain subunit proteins prepared by gene engineering, and adjuvants such as Freund's adjuvant, Cholera toxin(CT),E.coli heat labile toxin(LT) and lipid A, all of which may just non-specifically potentiate the immune responses. On the other hand, although the traditional non-living whole-cell vaccines usually have higher efficiency, current methods to inactivate infectious agents generally reduce or alter the vaccine's antigenicity, like heat-killing, irradiation or chemical treatment. As a novel vaccine or antigen delivery system, "Bacterial ghost"is a kind of empty bacterium which loses its cytoplasm and DNA while only remaining outer membranes. It is produced by controlled expression of cloned bacteriophage PhiX174 lysis gene E in bacterium. It has been shown that the protein coded by E gene leads to the formation of a transmembrane tunnel structure through which the components of the cytoplasm are expelled completely. Because the resμlting bacterial ghosts share all functional and antigenic determinants of the envelope with their living counterparts, they can target antigen to the musocal tissue and easily be captured by the antigen presenting cells(APCs). On the other hand, the prerequisite of H.pylori colonization and pathogenesis is its attachment to the epithelial cells of stomach. It is supposed to efficiently prevent and cure the infection of H.pylori by blocking its attachment. In this case, Hp adhesion A (HpaA) has been shown to be the main adhesion factor of H.pylori as well as a Hp unique antigen. The amino acid sequence of HpaA is highly conserved, and HpaA specific antibody can be detected in most patients with H.pylori infection. Therefore, HpaA is a good antigen candidate for developing H.pylori vaccine. In the present study, the major aim is to construct recombinant E.coli ghost expressing HpaA as a novel Hp vaccine, i.e. using bacterial ghost of E.coli as an antigen delivery system or an inner adjuvant for HpaA antigen. For this aim, HpaA antigen was firstly expressed and achored to the cytoplasmic membrane of E.coli, followed by induction of bacteria lysis to produce recombinant E.coli ghost. Based on the successful preparation of E.coli ghost, two technical strategies were carried out to prepare recombinant E.coli ghost expressing HpaA.One way is "coexpression of two plasmids".E.coli BL21(DE3) was cotransformed with plasmid pHH43 containing a lysis gene cassette and plasmid pBIOEX-E'-hpaA containing a hpaA expression cassette.Then, HpaA was induced to express with IPTG before E lysis gene expression by the heat induction. Another way is "one plasmid for two expressions",in which both of lysis gene cassette and hpaA expression cassette were constructed into the vector pET-28a,followed by IPTG and heat induction to produce recombinant E.coli ghost. The major results of this study are summarized as the following: 1.The preparation and identification of E.coli ghost. The plasmid pMuH36,containing E lysis gene cassette, was transformed into E.coli DH5αby CaCl2 method, and the expression of E lysis protein was induced by upregulating temperature.During the lysis procedure, the OD600 values were measured to obtain a lysis curve.The lysis rate was calculated by CFU(Colony Forming Unit). The morphology of E.coli ghost was identified by TEM(transmission electron microscope) and SEM(scanning electron microscope) . The results showed that E.coli ghost was successfully prepared with intact outer membrane of E.coli and the lysis rate was 95.07%, providing a good basis for the following studies. 2. The cloning and expression of HpaA in vector pET28a and pBIOEX. The membrance anthoring sequence E',1-54aa of E lysis protein,was amplified from plamid pMuH36 through PCR,and cloned into the plasmid pET28a-ltB-hpaA.Then,the recombinant expression plasmid pET28a-E'-hpaA was transformed into E.coli BL21(DE3)and induced with IPTG to express E'-HpaA protein, which was identified by PAGE, western-blotting. Thereafter, E'-hpaA segment was amplified from pET28a-E'-hpaA through PCR and subcloned into plasmid pBIOEX which is consistent with the plasmid of ColE1 origin. The recombinant pBIOEX-E'-HpaA was transformed into E.coli BL21(DE3)and induced with IPTG to express E'-hpaA protein ,which was examined by PAGE, western-blotting and ELISA. As a result, recombinant plasmid pET28a-E'-hpaA and pBIOEX-E'-HpaA wassuccessfully constructed, and could be induced to express target protein HpaA in E.coli. 3.The preparation of recombinant E.coli ghost by "coexpression of two plasmids"strategy.After cotransforming BL21(DE3)with the lysis plasmid pHH43 and consistent plasmid pBIOEX-E'-hpaA ,protein HpaA was firstly induced to express with IPTG and then E lysis protein was induced by temperature shift. To obtain an ideal condition to prepare recombitant E.coli ghost, the lysis rates were compared in different induction conditions and the expression level of HpaA were further examined by ELISA and immunofluorescence. Finally, an good preparation condition was decided.When bacteria was grown with OD600 up to 0.4, 1mmol/L IPTG was added to the growing culture .After 60min of the HpaA induction, cell lysis was induced by temperature shift. The lysis rate was proved to be 97.23%. 4. The preparation of recombinant E.coli ghost by "one plasmid for two expressions"strategy. The E lysis gene cassette from plasmid pHH43 was cloned into pET28a-E'-hpaA to obtain recombinant plasmid pET28a-hpaA-E-box, which was transformed into BL21(DE3)to produce the recombinant E.coli ghost in the same way. The results showed that the ideal condition to prepare this type of recombinant ghost was that when bacteria was grown with OD600 up to 0.4, 1mmol/L IPTG was added to the growing culture .After 60min of the HpaA induction, cell lysis was induced by temperature shift. The lysis rate was proved to be 98.57%. 5. Immunofluorescence of recombinant ghosts through the above two strategies showed that the rHpaA has successfully anchored to the membrane of the ghosts. And the TEM proved that most of recombinant ghosts were emptied with intact outmembrane structure. Moreover, Intraperitoneal immunization with these recombinant bacterial ghosts induced an HpaA -specific antibody response in BALB/c mice. As a conclusion, the present study successfully prepared the recombinant E.coli ghost expressing the HpaA,establishing the preliminary technique to prepare and identify this novel vaccine.This work may provide a good basis to develop an effective HP vaccine in the form of"Bacterial ghost".However, there still exist many disadvantages with the ghost system in this study, such as the low level of HpaA expression and immune response. We need following works to further optimize this system.