Study On Bacterial Ghosts Of Haemophilus Parasuis Based Phix174 Gene E Lysis Cassette-mediated

Bacterial ghost(BG)is a novel vaccine delivery system endowed with intrinsic adjubant properties.BGs are nonliving Gram-negative bacterial cell envelopes wh-ich are devoid of their cytoplasimic contents and DNA,yet maintain their cellular morphology and antigenic structures.It is produced by controlled expression of cloned bacteriophage PhiX174 lysis gene E in gram-negative bacterium.So the res-ulting bacterial ghosts share all functional and antigenic determinants of the enve-lope with their living counterparts.Therefore,it is significnce that BG was used in the researching of the animal vaccine to the animal immunity and introduce safe and efficiency novel vaccine.Expression of PhiX174 gene E cloned on a plasmid vector can be placed under transcriptional control of either the thermosensitiveλpL/pRcI857,lacpo-lacI8,under the lac -lacI promoter repressor systems,or the tol expression system.A major break through in temperature control of E-mediated lysi -s has been achieved by mutating the LambdaλpR gene expression regulation circu-it extending the heat stability of theλpR promoter/cI857 repressor system.The mut-atedλpR promoter/operator region resulted in new expression systems which stably repress gene E expression at temperatures up to 37℃,but still allow induct- ion of cell lysis at a temperature range of 39～42℃.The major results of this study are summarized as the following;1.The plasmid pHH43,containing E lysis gene cassette,was transformed into E.coli DH5αby CaCl₂ method,and the expression of E lysis protein was induced by upregulating temerature.The lysis rate was calculated by CFU(Colony Forming Unit), and the lysis rate was 99.67%.According to the SEM(scanning electron microscope) photograph,a lysis hole appeares in the middle or two poles of the cell.But this plasmid was failed to transformed into the Haemophilus parasuis.2.PhiX174 gene E cassette was coloned into the plasmid pLS88,construct the pLS88-E.The digesting and sequencing result is identical with the report outcome.3.The plasmid pLS88-E was transformed into the E.coli and Haemophilus parasuis, induced lysis procedure and compare the lysis rate.the result indicated that the concentration of the bacterial and living bacteria in the cultivate flat descend greatly;Haemophilus parasuis lysis rate was 99.84%,but only 82.04% in the E.coli.. in the SEM photograph,we find hole in the cell membranes.Thus it can be proved that the Haemophilus parasuis ghost was successful prepared.As a conclusion,this work may provide a technique basis to develop an effective Haemophilus parasuis and other gram-negative bacterial vaccine in the form of bacterial ghost.