Researches On Plasmid-independent Bacterial Ghost Vaccine And Chinese Herbs As Its Adjuvant

Bacterial ghosts are generated by the controlled exprssion of the PhiX174 lysis gene E in Gram-negative bacteria,which loss cytoplasm,nucleic acid,ribosomes and the capacity of reproduction.As a result,bacterial ghosts are bacterial cell envelops inactivated without denaturing process,hence retain all antigenic determinants as the same as their living counterparts and thus represent better killed vaccine candidates than traditional bacterin inactivated with formalin.On the base of bacterial ghost preparation tecnology depending on plasmid-borne lysis gene E,we constructed a bacterial ghost strain of Avian Pathogenic Escherichia coli(APEC)by means of homologous recombinant,in which the expression cassette of lysis gene E was integrated into the chromosome of the bacteria.This plasmid-independent bacterial ghost technology could overcome the insufficiency of lysis efficiency resulted from the loss of lysis plasmids in the preparation of bacterial ghosts with traditional plasmid-depenent bacterial ghost technology.On the other hand, antibiotics used in traditional plasmid-depenent bacterial ghost technology as selction pressure is no longer necessary,which in turn not only lower the cost of bacterial ghost preparation but also avoid the risk of antibiotic abuse.Thus,this plasmid-independent bacterial ghost technology has promising perspective of application.Traditional Chinese herbs are characterized with low toxicity and less adverse effect. Additionally,many of them have been found to function in adjusting immunity of animals in many ways.In this study,Yupingfeng injection was prepared and evaluated as immunoadjuvant of bacterial ghost vaccine in chicks.The main contents of the research are as follows:1.Construction of plasmid pTLacZ:LCLacZ gene was amplified with PCR from Avian Pathogenic Escherichia coli(APEC)wild strain RC-123,cloned into pMD18-T vector to construct plasmid pTLacZ.The lysis gene E expression cassette(LC)was amplified with PCR from the broad-host-range lysis plasmid pBBRlys,cloned into pMD18-T vector to construct plasmid p-T-LC.Plasmid p-T-LC and pTLacZ were cut by the same restriction enzyme BssHâ…¡,and then were ligated by T4 DNA ligase to construct plasmid pTLacZ:LC.2.Construction of plasmid-independent bacterial ghost strainThe allelic fragment LacZ:LC was amplified with PCR from plasmid pTLacZ:LC,and then electroporated into competent cells of APEC wild strain RC-123.Allelic exchange between allelic fragment LacZ:LC and LacZ gene in chromosome,of RC-123 via homologous recombination took place and resulted in integration of LC into LacZ gene in chromosome of RC-123.Mutant clone showed as white colony because of insert inactivation of LacZ gene on plate containing X-gal was plasmid-independent bacterial ghost strain and named as RC-123(LC).3.Dynamic tests of bacterial strainsGhosts of RC-123(LC)and RC-123(pBBRlys,plasmid-dependent bacterial ghosts strain)were generated successfully by increasing the incubation temperature from 28℃up to 42℃to induce the expression of lysis gene E.Compared with RC-123(pBBRlys),whose lysis was observed to begin at about 45～60 min.and complete within 90 min.after in duction of gene E expression,the onset of lysis of RC-123(LC)occurred 60～75 min.after temperature elevation and the lysis process completed within 105 min.At the end of the lysis process,the inactivation efficiency of RC-123(LC)and RC-123(pBBRlys)ghosts were determined as 99.9992%and 99.9916% respectively by plating and CFU counting.However,no living cell was detected in lyophilized RC-123(LC)and RC-123(pBBRlys)ghosts.4.Preparation of Yupingfeng injection4 Chinese herbs Milkvetch Roo,Largehead Atractylodes Rhizome and Divaricate Saposhnikovia Root according to the proportion of the recipe of Jade Screen Powder were used to prepare Yupingfeng injection by means of water and alcohol extraction method.Yupingfeng injection preparation(1g/ml)showed as clear,brownish red to brown aqueous liquid(PH 4.0～5.5). All technical indexes meet the requirements in Pharmacopeia.Seven-day-old chicken were intramuscularly injected with 0.8 ml(4 folds of normal dosage),no toxic and other adverse effects were observed.5.Immunoprotection testsChicken were intramuscularly vaccinated with RC-123(LC)ghosts and the traditional formalin killed RC-123 vaccine(FKV)respectively to compare the effectiveness of immunoprotection.At the same time,Yupingfeng injection was used as adjuvant of bacterial ghost vaccine,Adjuvant groupâ… were injected intramuscularly with Yupingfeng injection twice at the same time with vaccination and 1 day before respectively,while adjuvant groupâ…¡were injected with Yupingfeng injection only once when they were vaccinated.The result showed that RC-123(LC) ghost vaccine group showed a RPS of 77.78%after intramuscularly challenged with homologous RC-123 wild strain with a dose of two fold of LD₅₀,and its PRS was higher than that of the FKV group(64.89%).Additionally,the PRS of Yupingfeng adjuvant groupâ… (88.89%)andâ…¡(83.33%) are both higher than that of the groupe without adjuvant. The result of our study suggests that bacterial ghost is a promising vaccine candidate,which potentially has better protective efficiency than traditional formalin killed bacterial vaccines. Furthermore,Yupingfeng injection made from Chinese herbs is proved to have evident adjuvant effect.