Construction Of The Prokaryotic Expression Vecter With Lysis Gene E And SN Gene And Preparation Bacteria Ghost Of Escherichia Coli O₁₃₈

Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines.Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines. Colibacillosis is a kind of important zoonosis. It is susceptivity especially in the newborn domestic animals. It can cause growth retardation, low productivity and even death. Escherichia coli O138 is one of the important zoonosum bacteria causing Edema disease .The traditional vaccine of of swine edema disease which was widely used in hoggerys was developed on the base of O antigens,new genetic vaccine had to additional adjuvant to evocating non-specificity immune response.To protect swines out of edema disease, a new kind of Edema disease vaccine E. Coli O138 bacterial ghosts was productedTo construct the coexpression vector for PhiX174 lysisi gene E and Staphylococcus nuclease to prepare highly purified Escherichia coli bacterial ghosts,a pair of primers was designed according to the lysis gene E nucleotide sequence. With the primers, the whole segment of lysis gene E was amplified by PCR from plasmid PhiX174 RFI DNA which contains the full length lysis gene E, and then inserted into plasmid pBV-220 to generate a prokaryotic expression plasmid pBV-E. Based on the lysis gene E ,the expression of a secondary killing gene Staphylococcus nuclease A (SN) was suggested to give rise to the complete genetic inactivation of the bacterial samples. The conjunction gene was also inserted into recombinant plasmid thermosensitive plasmid pBV-220 to generate prokaryotic coexpression plasmids pBV-E-5L-SN,pBV-E-15L-SN .The recombinant plasmids was transferred into the Escherichia coli DH5αby CaCl2 to product bacterial ghosts after increasing the tempreture into 42℃. Lysis gene E and the conjunction genes were also successfully inserted into the recombinant vecter pGEX-6p-1,constructed E.coli bacterial ghosts expression vecter pGEX-E,pGEX-E-5L-SN,pGEX-E-15L-SN.The recombinant plasmids was transferred into the E. coli BL21(DE3) by CaCl2 to product bacterial ghosts after inducing with IPTG.Both of the thermosensitive and recombinant conjunction vecters were transferred into the E. coli O138 producing bccterial E. coli O138 ghosts.TEM observation proved that most of the lysed bacteria were emptied , whereas the whole outmembrane structur was no difference from the bacteria which nonlysed.The coexpression vector of PhiX174 lysisi gene E and Staphylococcus nuclease A was successfully constructed, and highly purified E.coli O138 bacterial ghosts was obtained. Immunization of mice with E. coli O138 ghosts without the addition of any adjuvant induced cellular and humoral immunity ,this indicate that it is possible to develop bacterial ghost vaccine.