Construction Of Recombinate Low-toxic Lipopolysaccharidesproducing Escherichia Coli J5 Mutant And Identification Of Relevant Products

Lipopolysaccharide(LPS)is the main component of the cell wall of gram-negative bacteria and plays important roles in bacterial survival and growth.On the one hand,LPS could stimulate host immune system and enhance the immunity.On the other hand,it owns strong cytotoxicity which is easy to damage to host.Monophosphoryl lipid A(MPLA)is one of the major derivative of lipid A,which has very good immune adjuvant function.Nowadays,commercial MPLA mainly extracts from LPS of Salmonella by chemical means called phenol-chloroform-petroleum ether(PCP)method,and then removes the phosphoric group through acidification.However,the yield of refined MPLA is low and the process is so complex which means high costs.Otherwise these refined LPS still contained inorganic ions such as Ca+ and Mg2+ and polyamines with positive charge,resulting in reduced solubility of LPS.Meanwhile,MPLA production from Salmonella,which is an important zoonotic pathogen,may be contaminated and affect the public health.In this study,E.coli J5 vaccine strain with natural incomplete o-antigen was selected as the original starting strain.IpxE gene of F.novicida and lpxM gene of E.coli are knock in and knock out,respectively,by using ?-Red homologous recombination technology,that transfer the strain J5 to an engineered strain producing MPLA with a low-toxicity The transformation process is as follows:Primers were designed to amplify lpxE segments and cat segments,which will be further used to replace the lacI in choromsome of J5.SOE-PCR was done combining the lpxE and cat together with homologous arm of lacl at the distal end.With ? Red-mediated recombination system,the PCR products were introduced into J5 strain carrying a heat-sensitive plasmid pKD46 by electroporation,and lacI gene was replaced by lpxE-cat fragment.The successful recombinant J5?lacI::lpxE-cat strains were selected by LB plates containing chloramphenicol and confirmed by PCR.Then the chloromycin-resistance gene was eliminated by another heat-sensitive plasmid pCP20,which encoding the Flp recombinase.Finally,the isogenic J5?lacI::lpxE mutants were confirmed by PCR and subsequent DNA sequencing.Similarly,according to lpxM gene sequence published in Genbank,primers were designed and J5?lacI::lpxE?lpxM mutant was constructed in a same way.In this study,hydrothermal phenol method was performed to extract LPS from E.coli DH5a,J5 strain andJ5?lacI::lpxE?lpxMmutant.The extracted LPS was further purified by adding DNase and RNase,and the quality was identified by silver dyeing.The results showed that the nucleic acid and protein residues in extracted LPS were little,which would not affect the subsequent experiments.Tachypleus Amebocyte Lysate analysis revealed that,LPS activity of DH5a was 2.3×105 EU/mg,while of J5 and J5?lacI::lpxE?lpxM were 1.7×105 EU/mg and4.3×103 EU/mg,respectively.LPS activity of J5?lacI::lpxE?pxM mutant was significantly lower than that of DH5? and J5 original bacteria.4 weeks old ICR mice were intraperitoneally injected with LPS extracts,and physiological and mental state and symptoms of the challenged mice were recorded.Once the mice were dissected,liver was taken out to make paraffin sections,which helping determine the toxicity of different LPS extracts.The results showed that,mice challenged with LPS extracts of J5 and DH5a showed obviously toxic response,behaving clothing hair mess and mental depression.The LPS extract of J5?lacI::lpxE?lpxM and PBS challenged mice were normal without any clinical symptoms.In summary,we sucessfully constructed the J5?lacI::lpxE?lpxM mutant producing low-toxicity LPS in this study.The low-toxicity MPLA productiongenerated by this strian,will improved the current production process of MPLA as well as significantly reduce its production costs.Application of this cheap and efficient MPLA adjuvant in veterinary vaccine would lay a basis for the development of new veterinary adjuvant.